Wednesday, December 22, 2010
Tuesday, December 14, 2010
Sunday, November 28, 2010
Hybridization techniques
HYBRIDIZATION TECHNIQUES
The genomes of most organisms contain the essential information which contributes to its various features and characteristics. The sequence of nucleotides that contributes to a particular biological characteristic is like a molecular signature and will e detectable if appropriate techniques are developed.It is here that nucleic acid hybridization is of great help to us.
The information of the double helix from 2 complementary strands of nucleic acids is the basis of nucleic acid hybridization technique.This procerss was first discovered by MARMUR and DOTY in 1961 .Two important features of this process were very soon established , the two sequences involved in duplex formation must have a degree of complemantarity , and the stabilty of the duplex formed depends on the extent of complementarity .
Nucleic acid hybridization is of central importance to genetic engeneering and is finding increasing use in molecular biology.Nucleic acid hybridization is the basis for rapid and realible assays developed for molecular characterization.The physical basis of these systems is precise nucleotide base paring and hydrogen bonding between two complementary strands of nucleotide sequence.Techniques of nucleic acid hybridization is developed from classical experiments of Gillespie and Speigelman.
TYPES OF HYBRIDIZATION:
1)Solution hybridization
2)Filter hybridization
3)Polymerase chain reaction
4)in Situ hybridization
5)Hybridization to 'chips'.
SOLUTION HYBRIDIZATION:
In this method,the reacting species(denatured,single strands of nucleic acids) are free in solution and are incubated under conditions that favour hybrid formation.To detect hybrid formation, advantage is taken of the difference in physical properties between single and double stranded nucleic acids.Duplex formation is usually measured by hypochromicity(i.e,reduction in the absorbance at 260nm as double standard reagions are formed) or selective binding of single or double strands to hydroxypatite columns.
USES:
a)To find out number of copies of sequences and their relatedness.The degree of relatedness between sequences can be analysed by measuring the Tm of reassociated DNA.
b)To determine wheather DNA samples contain sequences that are repeated relative to others..This is based on the principle that sequences present at higher concentration reassociate faster than those at lowr concentration.
c)Size of genome can be deduced.
d)Reapeted and single copy sequences can be isolated.
e)Number of different species of RNA in a cell and the number of copies of each species can be determined.
f)The proportion of DNA that is transcribed into mRNA can be derived from saturation hybridization studies.
g)The degree of overlap of RNAs expressed in different cell types can be measured.
h)It can also be extensively used for mapping the termini of transcripts in studies of gene organisation.
FILTER HYBRIDIZATION:
Denatured DNA or RNA is immobilised on inert support like nitrocellulose membrane, in such a way that self annealing is prevented,yet bound sequences are available for hybridization with added nucleic acid probe.To facilitate the analysis , the probe is labelled.Detection of hybrids is through detection of probe.
Filter hybridization is a versatile technique with convenience for large number of samples.It can be used quantitatively in conjunction with a calibration curve for estimating copy number of sequences.But it is most commonly used semiquantitatively to compare relative amount of sequences in differeny samples.
The main disadvantage of filter hybridization is that it is much slower than solution hybridization and time required for hybridization to go to completion is very long.therefore this technique is less useful than solution hybridization for analysing rare sequences.
For convenience sake filter hybridization is divided into primary and secondary techniques.
a)In primary screening, colonies of bacteria transformed with recombinant plasmids or bacteriophage plaques are replicated in Situ on nitrocellulose filter.DNA liberated is tested.
EXAMPLE:Colony hybridization,Plaque hybridization etc.
b)In secondary screening, DNA prepartions are boumd to inert support and studied
eg:Dot blot,southern blot etc.
USES:
a)Filter hybridization is capable of great discrimination and can detect single base changes in nucleic acid.This application is widely used in medical research, to detect mutations that cause disease.
b)To isolate phage and plasmids containing sequences of intrest from recombinant libraries.
In Situ HYBRIDIZATION:
This method is powerful and is used to locate nucleic acid sequences in histological and cytological preparations of tissues, organelles, cells and chromosomes.Samples are pretreated before hybridization .Car should be taken to retain morphological features of tissues or chromosome to avoid extraction or modification of nucleic acid, to avoid change in localization of nucleic acid and possible access of probes and detection reagents to nucleic acid.
USES:
a)To identify the location of genes on normal and abberent chromosomes
b)To identify the sites og gene expression
c)To determine level of trancription and changes in it along with development.
d)To detect chromosomal translocations in residual disease.
DOT BLOTS(SPOT BLOTS) (SLOT BLOT):
This si the simplest type of hybridization analysis.It measures the abundance of target sequences in a sample.Nucleic acid samples are not subjected to electrophoresis, but are spotted on to filters and hybridization is carried out as Northern or Southern blots.The technique is used for obtaining quantitative data in study of gene expression .The technique is rapid and simultaneous screening of many samples can be done.The technique is very sensitive and usesradioactive probes of high specific activity.It can detect as little as 1 pg of target in overnight exposure of hybridized filter.Dot blots are less efficient than southern blots in discrimination between correct hybridization and cross hybridization.The signal in dot blot is sum of all hybridizing species in a sample while in southern blot only strongly hybridizing material can be picked out of backgrouns smear.
Commercial apparatus has been developed for binding multiple samples of DNA to filters.Manual application is possible but is more time consuming and also dots are less uniform than those obtained with device.Results however are satisfactory.
protocols develpoed may fall into 2 classes:DNA denatured before or after its application to the filter. Both give satisfactiory results.
To common ways of obtaining linear or nicked DNA are- enzymatic digrstion or treatment at high temperature.The later partially depurinates the DNA so that on subsequent treatment with alakli the phosphodiester bond breaks at the sites of depurination.Linear DNA will then seperate into single strands.
For southern , northern and Dot blots both nitrocellolose and nylon filters give excellent results.
Nitrocellulose filter is usually treated with high concentration of salts either at the same time as , or prior to, binding of nucleic acids.This improves the efficiency of binding and Dot remain small.
STEPS OF DOT BLOTTING:
1)DNA is denatured by boiling the sample.This treatment partially depurinates the DNA.
2)Nitrocellulose or nylon or positively charged nylon filters are used for dot-blot analysis.For small amounts of nucleic acid in the sample, activated papers may be used..Positively charged nylon membranes bind DNA covalently at high pH.Samples for dot and slot blotting can therefore be applied in alkaline buffer which promote both denatured of DNA and binding to the membrane.
3)Sample as treated in step 1 are applied as dots on filter.
4)After appling all samples alkali treatment is given to filter.Samples are fully denatured on filter.Alkali treatment assists is nicking and linearising of supercoiled plasmid DNA .It also breaks phosphodiester bonds to break at site of depurination.
5)Filter is quickly neutralised and DNA is immobilised by baking at 80 degree centigrade -2hrs or by UV linking
6)32P labelled probes can detect 1-5 pg of target DNA.
In overnight exposure approximately 10-100 pg of target DNA gives easily identifiable signals.Single copy sequence of 1 kb represents 3pg of 10mug sample of human DNA If target sequence has more copies then DNA used for loading can be smaller.
Dot hybridization ios more sensitive than colony hybridization.Although both work on same principle dot blot is more laborious as it requires initial isolation of plasmid DNA from each clone.The plasmid may be extracted by one of the rapid 'mini' plasmid preparation and then DNA is spotted on nitrocellulose filters and hybridization is carried out.
ADVANTAGES OF DOT BLOT:
1)Technique is rapid .
2)Simultaneous screening of many samples can be done.
3)It detects as little as 1pg of target DNA.
4)Highly sensitive
5)Quqantiyative data in gene expression obtained.
6)Filter bound sequences can be analused under different conditions.
DISADVANTAGES OF DOT BLOTS:
1)It cannot differentiate between corect hybridization and cross hybridization.
2)More laborious as it requires initial isolation of plasmid DNA.
APPLICATION OF DOT BLOT HYBRIDIZATION:
1)The technique is qualitative and can distinguish between closely related members of multigene families and between sequences thet differ by single nucleotide .This property can be exploited in detection of mutations in prenatal diagnosis of genetic disease.
2)The technique can be used as semi quantitative method for estimating the relative levels of sequences in different samples.
COLONY HYBRIDIZATION:
Grunstein and Hogness developed an in Situ method for detection of recombinant clones.Modification introduced by Thayer are claimed to increasethe sensitivity and reproducibility of the technique by improving efficiency of cell lysis and immobilisation of DNA.
Success of colony hybridization depends on high specificity of synthetic oligonucleotide probe used.
APPLICATIONS:
1)food samples may be tested for organisms containing particular gene.20-100 cells is the lower limit of detection.Detection of pathogen in food samples can be done..Colony hybridization can be quantitative use but it is labour intensive.
2)Colony hybridization can be used for screening for genomic library or cDNA library and to isolate specific DNA sequence,Here library exists in the form of mixture of bacteria transformed with chimeric DNA molecules.Specific DNA sequence may be isolated using corresponding probe and colony hybridization technique.
DIP STICK:
Colony hybridization technique can be quantitative use but is labour intensive.A two probe system can be developed that uses a dip stick to remove the probe target complexes from hybridization reaction mixtue where both target, and probe are free in solution .This technique uses non-radioisotopic labelling system and is well suited for analysis of large number of food samples after completion of appropriate enrichment scheme.The test is qualitative in nature.Gene probes targeted to regiong of robosomal RNA are used and can detect a single cell in 25gms of sample.
PLAQUE HYBRIDIZATION:
Benton Devis in 1977 developed the modification of Grunstein and Hogness method to apply it to phage plaques.
When genomic or cDNA library is not available in the form of bacteria transformed with chimeric DNA molecules ,colony hybridization technique cannot be used for screening sequences.If genomic or cDNA library is available in the form of chemeric phage particles carrying cloned segments,then plaques formed by such phages have to be screened.These plaques are treated judst like colonies in colony hybridization to identify and isolate chimeric phage particles carrying the gene of intreast. This technique is then discribed as ;plaque hybridization'.
Nitrocellulose paper is applied to upper surface of agar plates making direct contact between plaques and filter.Plaques contain consider amount of unpacakaged recombinant DNA. This then binds to filter and be denatured ,fixed and hybridized.
The genomes of most organisms contain the essential information which contributes to its various features and characteristics. The sequence of nucleotides that contributes to a particular biological characteristic is like a molecular signature and will e detectable if appropriate techniques are developed.It is here that nucleic acid hybridization is of great help to us.
The information of the double helix from 2 complementary strands of nucleic acids is the basis of nucleic acid hybridization technique.This procerss was first discovered by MARMUR and DOTY in 1961 .Two important features of this process were very soon established , the two sequences involved in duplex formation must have a degree of complemantarity , and the stabilty of the duplex formed depends on the extent of complementarity .
Nucleic acid hybridization is of central importance to genetic engeneering and is finding increasing use in molecular biology.Nucleic acid hybridization is the basis for rapid and realible assays developed for molecular characterization.The physical basis of these systems is precise nucleotide base paring and hydrogen bonding between two complementary strands of nucleotide sequence.Techniques of nucleic acid hybridization is developed from classical experiments of Gillespie and Speigelman.
TYPES OF HYBRIDIZATION:
1)Solution hybridization
2)Filter hybridization
3)Polymerase chain reaction
4)in Situ hybridization
5)Hybridization to 'chips'.
SOLUTION HYBRIDIZATION:
In this method,the reacting species(denatured,single strands of nucleic acids) are free in solution and are incubated under conditions that favour hybrid formation.To detect hybrid formation, advantage is taken of the difference in physical properties between single and double stranded nucleic acids.Duplex formation is usually measured by hypochromicity(i.e,reduction in the absorbance at 260nm as double standard reagions are formed) or selective binding of single or double strands to hydroxypatite columns.
USES:
a)To find out number of copies of sequences and their relatedness.The degree of relatedness between sequences can be analysed by measuring the Tm of reassociated DNA.
b)To determine wheather DNA samples contain sequences that are repeated relative to others..This is based on the principle that sequences present at higher concentration reassociate faster than those at lowr concentration.
c)Size of genome can be deduced.
d)Reapeted and single copy sequences can be isolated.
e)Number of different species of RNA in a cell and the number of copies of each species can be determined.
f)The proportion of DNA that is transcribed into mRNA can be derived from saturation hybridization studies.
g)The degree of overlap of RNAs expressed in different cell types can be measured.
h)It can also be extensively used for mapping the termini of transcripts in studies of gene organisation.
FILTER HYBRIDIZATION:
Denatured DNA or RNA is immobilised on inert support like nitrocellulose membrane, in such a way that self annealing is prevented,yet bound sequences are available for hybridization with added nucleic acid probe.To facilitate the analysis , the probe is labelled.Detection of hybrids is through detection of probe.
Filter hybridization is a versatile technique with convenience for large number of samples.It can be used quantitatively in conjunction with a calibration curve for estimating copy number of sequences.But it is most commonly used semiquantitatively to compare relative amount of sequences in differeny samples.
The main disadvantage of filter hybridization is that it is much slower than solution hybridization and time required for hybridization to go to completion is very long.therefore this technique is less useful than solution hybridization for analysing rare sequences.
For convenience sake filter hybridization is divided into primary and secondary techniques.
a)In primary screening, colonies of bacteria transformed with recombinant plasmids or bacteriophage plaques are replicated in Situ on nitrocellulose filter.DNA liberated is tested.
EXAMPLE:Colony hybridization,Plaque hybridization etc.
b)In secondary screening, DNA prepartions are boumd to inert support and studied
eg:Dot blot,southern blot etc.
USES:
a)Filter hybridization is capable of great discrimination and can detect single base changes in nucleic acid.This application is widely used in medical research, to detect mutations that cause disease.
b)To isolate phage and plasmids containing sequences of intrest from recombinant libraries.
In Situ HYBRIDIZATION:
This method is powerful and is used to locate nucleic acid sequences in histological and cytological preparations of tissues, organelles, cells and chromosomes.Samples are pretreated before hybridization .Car should be taken to retain morphological features of tissues or chromosome to avoid extraction or modification of nucleic acid, to avoid change in localization of nucleic acid and possible access of probes and detection reagents to nucleic acid.
USES:
a)To identify the location of genes on normal and abberent chromosomes
b)To identify the sites og gene expression
c)To determine level of trancription and changes in it along with development.
d)To detect chromosomal translocations in residual disease.
DOT BLOTS(SPOT BLOTS) (SLOT BLOT):
This si the simplest type of hybridization analysis.It measures the abundance of target sequences in a sample.Nucleic acid samples are not subjected to electrophoresis, but are spotted on to filters and hybridization is carried out as Northern or Southern blots.The technique is used for obtaining quantitative data in study of gene expression .The technique is rapid and simultaneous screening of many samples can be done.The technique is very sensitive and usesradioactive probes of high specific activity.It can detect as little as 1 pg of target in overnight exposure of hybridized filter.Dot blots are less efficient than southern blots in discrimination between correct hybridization and cross hybridization.The signal in dot blot is sum of all hybridizing species in a sample while in southern blot only strongly hybridizing material can be picked out of backgrouns smear.
Commercial apparatus has been developed for binding multiple samples of DNA to filters.Manual application is possible but is more time consuming and also dots are less uniform than those obtained with device.Results however are satisfactory.
protocols develpoed may fall into 2 classes:DNA denatured before or after its application to the filter. Both give satisfactiory results.
To common ways of obtaining linear or nicked DNA are- enzymatic digrstion or treatment at high temperature.The later partially depurinates the DNA so that on subsequent treatment with alakli the phosphodiester bond breaks at the sites of depurination.Linear DNA will then seperate into single strands.
For southern , northern and Dot blots both nitrocellolose and nylon filters give excellent results.
Nitrocellulose filter is usually treated with high concentration of salts either at the same time as , or prior to, binding of nucleic acids.This improves the efficiency of binding and Dot remain small.
STEPS OF DOT BLOTTING:
1)DNA is denatured by boiling the sample.This treatment partially depurinates the DNA.
2)Nitrocellulose or nylon or positively charged nylon filters are used for dot-blot analysis.For small amounts of nucleic acid in the sample, activated papers may be used..Positively charged nylon membranes bind DNA covalently at high pH.Samples for dot and slot blotting can therefore be applied in alkaline buffer which promote both denatured of DNA and binding to the membrane.
3)Sample as treated in step 1 are applied as dots on filter.
4)After appling all samples alkali treatment is given to filter.Samples are fully denatured on filter.Alkali treatment assists is nicking and linearising of supercoiled plasmid DNA .It also breaks phosphodiester bonds to break at site of depurination.
5)Filter is quickly neutralised and DNA is immobilised by baking at 80 degree centigrade -2hrs or by UV linking
6)32P labelled probes can detect 1-5 pg of target DNA.
In overnight exposure approximately 10-100 pg of target DNA gives easily identifiable signals.Single copy sequence of 1 kb represents 3pg of 10mug sample of human DNA If target sequence has more copies then DNA used for loading can be smaller.
Dot hybridization ios more sensitive than colony hybridization.Although both work on same principle dot blot is more laborious as it requires initial isolation of plasmid DNA from each clone.The plasmid may be extracted by one of the rapid 'mini' plasmid preparation and then DNA is spotted on nitrocellulose filters and hybridization is carried out.
ADVANTAGES OF DOT BLOT:
1)Technique is rapid .
2)Simultaneous screening of many samples can be done.
3)It detects as little as 1pg of target DNA.
4)Highly sensitive
5)Quqantiyative data in gene expression obtained.
6)Filter bound sequences can be analused under different conditions.
DISADVANTAGES OF DOT BLOTS:
1)It cannot differentiate between corect hybridization and cross hybridization.
2)More laborious as it requires initial isolation of plasmid DNA.
APPLICATION OF DOT BLOT HYBRIDIZATION:
1)The technique is qualitative and can distinguish between closely related members of multigene families and between sequences thet differ by single nucleotide .This property can be exploited in detection of mutations in prenatal diagnosis of genetic disease.
2)The technique can be used as semi quantitative method for estimating the relative levels of sequences in different samples.
COLONY HYBRIDIZATION:
Grunstein and Hogness developed an in Situ method for detection of recombinant clones.Modification introduced by Thayer are claimed to increasethe sensitivity and reproducibility of the technique by improving efficiency of cell lysis and immobilisation of DNA.
Success of colony hybridization depends on high specificity of synthetic oligonucleotide probe used.
APPLICATIONS:
1)food samples may be tested for organisms containing particular gene.20-100 cells is the lower limit of detection.Detection of pathogen in food samples can be done..Colony hybridization can be quantitative use but it is labour intensive.
2)Colony hybridization can be used for screening for genomic library or cDNA library and to isolate specific DNA sequence,Here library exists in the form of mixture of bacteria transformed with chimeric DNA molecules.Specific DNA sequence may be isolated using corresponding probe and colony hybridization technique.
DIP STICK:
Colony hybridization technique can be quantitative use but is labour intensive.A two probe system can be developed that uses a dip stick to remove the probe target complexes from hybridization reaction mixtue where both target, and probe are free in solution .This technique uses non-radioisotopic labelling system and is well suited for analysis of large number of food samples after completion of appropriate enrichment scheme.The test is qualitative in nature.Gene probes targeted to regiong of robosomal RNA are used and can detect a single cell in 25gms of sample.
PLAQUE HYBRIDIZATION:
Benton Devis in 1977 developed the modification of Grunstein and Hogness method to apply it to phage plaques.
When genomic or cDNA library is not available in the form of bacteria transformed with chimeric DNA molecules ,colony hybridization technique cannot be used for screening sequences.If genomic or cDNA library is available in the form of chemeric phage particles carrying cloned segments,then plaques formed by such phages have to be screened.These plaques are treated judst like colonies in colony hybridization to identify and isolate chimeric phage particles carrying the gene of intreast. This technique is then discribed as ;plaque hybridization'.
Nitrocellulose paper is applied to upper surface of agar plates making direct contact between plaques and filter.Plaques contain consider amount of unpacakaged recombinant DNA. This then binds to filter and be denatured ,fixed and hybridized.
Wednesday, November 24, 2010
CORYNEBACTERIUM DIPHTHERIAE
Corynebacterim diphtheriae is a club shaped involution form or slender rod shaped(normal form), non motile, non capsulated, non sporing and gram positive bacteria. they are pleomorphic and measuring approximately 3-6muv m into 0.6-0.8muv.The bacilli may exist in pairs or groups.They commonly show chinese letter pattern(V, L, X)or cuniform arrangement.The cells shoe septa and 'polymeta phosphate granules' in the cytoplasm when stained with Loeffler;s methylene blue, the granules take up a bluish purple colour and hence they are called meta chromatic granules. They are also called volution or Babes Ernst granules.Diphtheria bacilli is classified into 3 types on the clinical severity.
1)Gravis
BIO-CHEMICAL REACTIONS:
Diphtheria bacilli ferment glucose,galactose,maltose and dextrin but not lactose , mannitol, or sucrose. On fermentation they produce acid but no gas.These are catalase positive.They reduce nitrates and donot hydrolyse gelatin.
RESISTANCE:
Diphtheria bacilli are destroyed by heat at 58 degree centigrade in 10 min and at 100 degerr centigrade in 1 min and are easily destroyed by antiseptics. They are susceptible to pencillin,erythromycin and broad spectrum antibiotics.
TOXIN:
Virulent strains of diphtheria bacilli produce a very powerful exotoxin and it is more antigenic. Production of diphtheria exotoxin is a result of tox* gene.The gene is carried by the bacteriophage is known as beta-phage.C.diphtheriae strains are lysogenic for this phage.
The diphtheria toxin is a protein and it has a molecular weight of about 62,000.It is extremely potent and the lethal dose for a 250g guinea pig is 0.0001mg. The toxin is labile and can be converted to toxoid at 37 degree centigrade for 4-6 weeks and treating it with 0.2-0.4% formalin.The diptheria toxin act by inhibiting protein synthesis in cardiac muscle, resulting in both structural and functional damage. Cardiac insufficiency can cause death.Demyelination caused by the toxin can affect both peripheral, cranial nerves and result in paralysis.
PATHOGENICITY;
The incubation period in diphtheria is commonly 3-4 days, but may on occasion be as short as one day.C.diphtheriae enters the body through respiratory track by inhalation.
The site of infection may be faucial, laryngeal, nasal, otitis, conjuctival, genital and cutaneous.After entry they establish in the upper respiratory track and multiply usually in the nose or throat.They have very little invasive ability and rarely enters into the blood or tissues and they release powerful exotoxin.Exotoxin consists of 2 fragments A and B. The 'B' fragment attaches to specific receptors on the cell membrane of the host cell and toxin is taken into the cell by endocytosis.
After the entry into the cell, the toxin is activated by protease,the 'A' fragment seperate from the 'B' fragment and becomes an active enzyme.This enzyme tranfers a protein of NAD of the cell on to an elongation factor -2(EF-2).which is essentially irreversible.It activates the EF-2 and there by stops protein synthesis.This causes local necrosis and th eresulting fibrinous exudate together with disintegrating epithelial cells, leucocytes and erythrocytes and bacteria formed a grey white pseudomembrane in the throat which is characteristic feature of diphtheria infection.
CLINICAL MANIFESTATIONS:
Sore throat, fever, fatigue, malaise, pseudomembrane formation on tonsils and throat, marked adenitis(bullneck), cellulitis, asphyxia, acute circulatory failure, otitis media, pneumonia,heart and kidney failure etc...
LABORATORY DIAGNOSIS:
Laboratory diagnosis consists of isolation od diphtheria bacillus and demonstration of its toxicity.
A)ISOLATION OF DIPHTHERIA BACILLUS:
a)MICROSCOPY: Specimens are collected from the throat swabs using tongue depressor and smear is preapred.After gram staining, it is observed under microscope for diphtheroids.The smear may also be stained with loeffler's methylene blue stain and observed under microscope.Its typical morphology identifies corynebacteria.
b)CULTUTE: For cluture, the swabs are inoculated on loeffers serum slope,potassium tellurite blood agar.On Loeffler's serum slope ,growth is observed within 6-8hrs and on potassium tellurite blood agar, grey or black colonies are observed.
B)DEMONSTRATION OF TOXICITY:
a)ELEK'S GEL PRECIPITATION TEST:
A rectangular strip of filter paper impreganated with diphtheria antitoxin (100units/ml)is placed on the surface of 20% normal horse serum agar in petridish.Testing strain is inoculated at right angle to filter paper strip and incubated at 37 degree centigrade for 24 -48 hrs.Toxin produced by bacterial growth will diffuse into agar and produce line of precipitation .They are compared with controls.No precipitate will occur in non toxigenic strain.
EPIDEMIOLOGY:
Diphtheria has been virtually erdicaated from most advanced countries.Thus in england and wales , between 1915 and 1942 , the number of diphtheri cases per year was about50,000 and deaths around 2,500-4,000.It was the commonest cause of death in children aged 4-10 yrs.It is rare in the first year of life, reaches a peak between 2 - 5 years, falls slowly between 5-10 years an drapidily between 10- 15 years.Infection is rare in early infany because of the passive immunity obtained from the mother.The disease is commoner in rural than in urban years. Carriers transmit the infection to their contacts.Fomites do not seem to play an important role though in special situations toys and pencils may act as vehicles of infection.
PROPHYLAXIS:
General control methods comprise early diagnosis, prompt hospitalization, recognition of carriers.Diphtheria can by a programme of mass imunisation , for this Diphtheria-Pertusis-Tetnus(DPT) vaccine is used.Booster immunisation at 10 year intervals maintain immunity.
The methods of immunisation available are active, passive or combined.Susceptibility to diphtheria can be detected by the schick test when the diphtheria toxin is injected intradermally into a susceptibility person , it causes a local reaction while in an immune individual, no reaction ensures as the toxin is neutralised by the antitoxin in blood circulation.
TREATMENT:
Specific treatment of diphtheria consists of antitoxic and antibiotic therapy.
C.diphtheria is sensitive to pencillin and can be cleared from the throat within few days by pencillin treatment.
1)Gravis
2)Intermedius
3)Mitis.
Diphtheria is a disease caused by the corynebacterium diphtheriae found in children.It is characterised by sore throat, fever, fatigue, malaise, pseudo membrane formation of tonsils and throat adenitis etc.
The disease was first recognised as a clinical entity by Bretonneau who called it 'diphtherite'.The name is derived from the tough, leathery, pseudo membrane formed in the disease.(Diphtheros means leather).Diphtheria bacillus was first observed and describad by klebs.It was cultivated by Loeffler bacillus.Roux and Yersin discovered diphtheria exotoxin and established its pathogenic effect.
CULTURAL CHARACTERISTICS:
Growth is scantu on the ordinart madia .Enrichment with blood, serum or egg is necessary for good growth.The optimum temperature for growth is 37 degree centigrade and optimum pH is 7.2. It is an aerobe and facultative anaerobe.On loeffler's serum agar medium, the bacteria out grows as small granular, moist, creamy colomies which gives a typical morphology. On potassium tellurite blood agar medium, they selectively grow and form black colour colonies.
BIO-CHEMICAL REACTIONS:
Diphtheria bacilli ferment glucose,galactose,maltose and dextrin but not lactose , mannitol, or sucrose. On fermentation they produce acid but no gas.These are catalase positive.They reduce nitrates and donot hydrolyse gelatin.
RESISTANCE:
Diphtheria bacilli are destroyed by heat at 58 degree centigrade in 10 min and at 100 degerr centigrade in 1 min and are easily destroyed by antiseptics. They are susceptible to pencillin,erythromycin and broad spectrum antibiotics.
TOXIN:
Virulent strains of diphtheria bacilli produce a very powerful exotoxin and it is more antigenic. Production of diphtheria exotoxin is a result of tox* gene.The gene is carried by the bacteriophage is known as beta-phage.C.diphtheriae strains are lysogenic for this phage.
The diphtheria toxin is a protein and it has a molecular weight of about 62,000.It is extremely potent and the lethal dose for a 250g guinea pig is 0.0001mg. The toxin is labile and can be converted to toxoid at 37 degree centigrade for 4-6 weeks and treating it with 0.2-0.4% formalin.The diptheria toxin act by inhibiting protein synthesis in cardiac muscle, resulting in both structural and functional damage. Cardiac insufficiency can cause death.Demyelination caused by the toxin can affect both peripheral, cranial nerves and result in paralysis.
PATHOGENICITY;
The incubation period in diphtheria is commonly 3-4 days, but may on occasion be as short as one day.C.diphtheriae enters the body through respiratory track by inhalation.
The site of infection may be faucial, laryngeal, nasal, otitis, conjuctival, genital and cutaneous.After entry they establish in the upper respiratory track and multiply usually in the nose or throat.They have very little invasive ability and rarely enters into the blood or tissues and they release powerful exotoxin.Exotoxin consists of 2 fragments A and B. The 'B' fragment attaches to specific receptors on the cell membrane of the host cell and toxin is taken into the cell by endocytosis.
After the entry into the cell, the toxin is activated by protease,the 'A' fragment seperate from the 'B' fragment and becomes an active enzyme.This enzyme tranfers a protein of NAD of the cell on to an elongation factor -2(EF-2).which is essentially irreversible.It activates the EF-2 and there by stops protein synthesis.This causes local necrosis and th eresulting fibrinous exudate together with disintegrating epithelial cells, leucocytes and erythrocytes and bacteria formed a grey white pseudomembrane in the throat which is characteristic feature of diphtheria infection.
CLINICAL MANIFESTATIONS:
Sore throat, fever, fatigue, malaise, pseudomembrane formation on tonsils and throat, marked adenitis(bullneck), cellulitis, asphyxia, acute circulatory failure, otitis media, pneumonia,heart and kidney failure etc...
LABORATORY DIAGNOSIS:
Laboratory diagnosis consists of isolation od diphtheria bacillus and demonstration of its toxicity.
A)ISOLATION OF DIPHTHERIA BACILLUS:
a)MICROSCOPY: Specimens are collected from the throat swabs using tongue depressor and smear is preapred.After gram staining, it is observed under microscope for diphtheroids.The smear may also be stained with loeffler's methylene blue stain and observed under microscope.Its typical morphology identifies corynebacteria.
b)CULTUTE: For cluture, the swabs are inoculated on loeffers serum slope,potassium tellurite blood agar.On Loeffler's serum slope ,growth is observed within 6-8hrs and on potassium tellurite blood agar, grey or black colonies are observed.
B)DEMONSTRATION OF TOXICITY:
a)ELEK'S GEL PRECIPITATION TEST:
A rectangular strip of filter paper impreganated with diphtheria antitoxin (100units/ml)is placed on the surface of 20% normal horse serum agar in petridish.Testing strain is inoculated at right angle to filter paper strip and incubated at 37 degree centigrade for 24 -48 hrs.Toxin produced by bacterial growth will diffuse into agar and produce line of precipitation .They are compared with controls.No precipitate will occur in non toxigenic strain.
EPIDEMIOLOGY:
Diphtheria has been virtually erdicaated from most advanced countries.Thus in england and wales , between 1915 and 1942 , the number of diphtheri cases per year was about50,000 and deaths around 2,500-4,000.It was the commonest cause of death in children aged 4-10 yrs.It is rare in the first year of life, reaches a peak between 2 - 5 years, falls slowly between 5-10 years an drapidily between 10- 15 years.Infection is rare in early infany because of the passive immunity obtained from the mother.The disease is commoner in rural than in urban years. Carriers transmit the infection to their contacts.Fomites do not seem to play an important role though in special situations toys and pencils may act as vehicles of infection.
PROPHYLAXIS:
General control methods comprise early diagnosis, prompt hospitalization, recognition of carriers.Diphtheria can by a programme of mass imunisation , for this Diphtheria-Pertusis-Tetnus(DPT) vaccine is used.Booster immunisation at 10 year intervals maintain immunity.
The methods of immunisation available are active, passive or combined.Susceptibility to diphtheria can be detected by the schick test when the diphtheria toxin is injected intradermally into a susceptibility person , it causes a local reaction while in an immune individual, no reaction ensures as the toxin is neutralised by the antitoxin in blood circulation.
TREATMENT:
Specific treatment of diphtheria consists of antitoxic and antibiotic therapy.
C.diphtheria is sensitive to pencillin and can be cleared from the throat within few days by pencillin treatment.
DNA SEQUENCING
DNA sequencing.
The term DNA sequencing refers to sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.
The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of dye-based sequencing methods with automated analysis, DNA sequencing has become easier and orders of magnitude faster.
History:
RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2, identified and published by Walter Fiers and his coworkers at the University of Ghent (Ghent, Belgium), between 1972 and 1976.Prior to the development of rapid DNA sequencing methods in the early 1970s by Frederick Sanger at the University of Cambridge, in England and Walter Gilbert and Allan Maxam at Harvard, a number of laborious methods were used. For instance, in 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis.
The chain-termination method developed by Sanger and coworkers in 1975 soon became the method of choice, owing to its relative ease and reliability.
Maxam–Gilbert sequencing:
In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases.
Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing,
Maxam–Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA. However, with the improvement of the chain-termination method (see below), Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity prohibiting its use in standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with scale-up.
The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first "cut" site in each molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA fragment, from which the sequence may be inferred.
Also sometimes known as "chemical sequencing", this method originated in the study of DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.
Chain-termination methods:
Part of a radioactively labelled sequencing gel
Because the chain-terminator method (or Sanger method after its developer Frederick Sanger) is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. The key principle of the Sanger method was the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.
The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence.
DNA fragments are labeled with a radioactive or fluorescent tag on the primer (1), in the new DNA strand with a labeled dNTP, or with a labeled ddNTP. (click to expand)
Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5’ end with a fluorescent dye. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.Sequence ladder by radioactive sequencing compared to fluorescent peaks (click to expand)
Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.Dye-terminator sequencing:
Capillary electrophoresis (click to expand)
Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths.Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis (see figure to the left).
This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.
Challenges:
Common challenges of DNA sequencing include poor quality in the first 15–40 bases of the sequence and deteriorating quality of sequencing traces after 700–900 bases. Base calling software typically gives an estimate of quality to aid in quality trimming.In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of the cloning vector. In contrast, PCR-based cloning and emerging sequencing technologies based on pyrosequencing often avoid using cloning vectors. Recently, one-step Sanger sequencing (combined amplification and sequencing) methods such as Ampliseq and SeqSharp have been developed that allow rapid sequencing of target genes without cloning or prior amplification.
Current methods can directly sequence only relatively short (300–1000 nucleotides long) DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide.
Automation and sample preparation:
View of the start of an example dye-terminator read (click to expand)
Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day. DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Sequencing reactions by thermocycling, cleanup and re-suspension in a buffer solution before loading onto the sequencer are performed separately. A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically. These programs score the quality of each peak and remove low-quality base peaks (generally located at the ends of the sequence). The accuracy of such algorithms is below visual examination by a human operator, but sufficient for automated processing of large sequence data sets.Amplification and clonal selection:
Genomic DNA is fragmented into random pieces and cloned as a bacterial library. DNA from individual bacterial clones is sequenced and the sequence is assembled by using overlapping DNA regions.(click to expand)
Large-scale sequencing aims at sequencing very long DNA pieces, such as whole chromosomes. Common approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA is cloned into a DNA vector, and amplified in Escherichia coli. Short DNA fragments purified from individual bacterial colonies are individually sequenced and assembled electronically into one long, contiguous sequence.This method does not require any pre-existing information about the sequence of the DNA and is referred to as de novo sequencing. Gaps in the assembled sequence may be filled by primer walking. The different strategies have different tradeoffs in speed and accuracy; shotgun methods are often used for sequencing large genomes, but its assembly is complex and difficult, particularly with sequence repeats often causing gaps in genome assembly.
Most sequencing approaches use an in vitro cloning step to amplify individual DNA molecules, because their molecular detection methods are not sensitive enough for single molecule sequencing. Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. Polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing.
Another method for in vitro clonal amplification is bridge PCR, where fragments are amplified upon primers attached to a solid surface, used in the Illumina Genome Analyzer. The single-molecule method developed by Stephen Quake's laboratory (later commercialized by Helicos) is an exception: it uses bright fluorophores and laser excitation to detect pyrosequencing events from individual DNA molecules fixed to a surface, eliminating the need for molecular amplification.
High-throughput sequencing:
The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once. High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS):
The first of the "next-generation" sequencing technologies, MPSS was developed in 1990s at Lynx Therapeutics, a company founded in 1992 by Sidney Brenner and Sam Eletr. MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences. Because the technology was so complex, MPSS was only performed 'in-house' by Lynx Therapeutics and no machines were sold; when the merger with Solexa later lead to the development of sequencing-by-synthesis, a more simple approach with numerous advantages, MPSS became obsolete. However, the essential properties of the MPSS output were typical of later "next-gen" data types, including hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels. Lynx Therapeutics merged with Solexa in 2004, and this company was later purchased by Illumina.454 pyrosequencing:
A parallelized version of pyrosequencing was developed by 454 Life Sciences. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other. 454 Life Sciences has since been acquired by Roche Diagnostics.Illumina (Solexa) sequencing:
Solexa, now part of Illumina developed a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification). One type of nucleotide at a time is then added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides and the dye is chemically removed from the DNA, allowing a next cycle.SOLiD sequencing:
Applied Biosystems' SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing.Future methods:
Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced. Mass spectrometry may be used to determine mass differences between DNA fragments produced in chain-termination reactions.DNA sequencing methods currently under development include labeling the DNA polymerase, reading the sequence as a DNA strand transits through nanopores, and microscopy-based techniques, such as AFM or electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording.
In microfluidic Sanger sequencing the entire thermocycling amplification of DNA fragments as well as their separation by electrophoresis is done on a single glass wafer (approximately 10 cm in diameter) thus reducing the reagent usage as well as cost. In some instances researchers have shown that they can increase the throughput of conventional sequencing through the use of microchips.Research will still need to be done in order to make this use of technology effective.
In October 2006, the X Prize Foundation established an initiative to promote the development of full genome sequencing technologies, called the Archon X Prize, intending to award $10 million to "the first Team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000 (US) per genome."
Monday, November 22, 2010
Polymerase chain reaction
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR
PCR principles and procedure
PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.
A basic PCR set up requires several components and reagents. These components include:
- DNA template that contains the DNA region (target) to be amplified.
- Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
- Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
- Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
- Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
- Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis
- Monovalent cation potassium ions.
Procedure
Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.
Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.
- Denaturation step:
- This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
- Annealing step:
- The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
- Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
- Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
- Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.
PCR stages
The PCR process can be divided into three stages:Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.Application of PCR
Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E. coli) can be rapidly screened by PCR for correct DNA vector constructs.PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.
Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing .This technique may also be used to determine evolutionary relationships among organisms.
Amplification and quantification of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar.Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.
PCR in diagnosis of diseases
PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques
Variations on the basic PCR technique
- Allele-specific PCR: a diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence
- Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product
- Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.[19] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
- Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation
- Hot-start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase.Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibodyor by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
- Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
- Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.
- .Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR
- Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
- Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
- Overlap-extension PCR: a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
- Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.
- Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).
- Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR(primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR(where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).
- Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.
- Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles
- PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.
- Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),5'RACE LaNe and 3'RACE LaNe
- History
The discovery in 1976 of Taq polymerase — a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments such as hot springs — paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle.This allowed an automated thermocycler-based process for DNA amplification.
When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car.He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat." He was awarded the Nobel Prize in Chemistry in 1993 for his invention,seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle (see below).
Patent wars
The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on March 28, 2005.[43]
Subscribe to:
Posts (Atom)