pcr types and applications
TRANSCRIPT
Polymerase Chain Reaction Polymerase Chain Reaction (PCR) and Its (PCR) and Its ApplicationsApplications
S.Karthikumar.,M.Sc.,M.Phil.,M.Tech.,(Ph.D)Assistant Professor, Department of Biotechnology
Kamaraj College of Engineering and Technology
Virudhunagar-626001, Tamilnadu, INDIA
What is PCR?What is PCR?PCR is an exponentially
progressing synthesis of the defined target DNA sequences in vitro.
It was invented in 1983 by Dr. Karry Mullis, for which he received the Nobel Prize in Chemistry in 1993.
Did He Really Invent PCR?
• The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971:– Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.
• Progress was limited by primer synthesis and polymerase purification issues.
• Mullis properly exploited amplification.
What is PCR? : What is PCR? : Why “Polymerase”?Why “Polymerase”?
It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
What is PCR? : What is PCR? : Why “Chain”?Why “Chain”?
It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
What is PCR? : What is PCR? : The “Reaction” ComponentsThe “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence to be amplified.
4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme [email protected] 6
The Basics of PCR Cycling
• 30–35 cycles each comprising:– denaturation (95°C), 30 sec.– annealing (55–60°C), 30 sec.– extension (72°C),
time depends on product size.
Taq polymerase
Thermus aquaticus, a thermophilic bacteria discovered in 1969 in
hot spring of Yellowstone National park . It can tolerate high temperature. The DNA polymerase (Taq polymerase) was isolated.
Thermostable PolymerasesPolymerase T ½,
95oC Extension
Rate (nt/sec) Type of
ends Source
Taq pol 40 min 75 3’A T. aquaticus
Amplitaq (Stoffel
fragment)
80 min >50 3’A T. aquaticus
Vent* 400 min >80 95% blunt
Thermococcus litoralis
Deep Vent* 1380 min ? 95% blunt
Pyrococcus GB-D
Pfu >120 min 60 Blunt Pyrococcus furiosus
Tth* (RT activity)
20 min >33 3’A T. thermophilus
*Have proof-reading functions and can generate products over 30 kbp
• Taq: Thermus aquaticus (most commonly used)– Sequenase: T. aquaticus YT-1
– Restorase (Taq + repair enzyme)
• Tfl: T. flavus• Tth: T. thermophilus HB-8• Tli: Thermococcus litoralis
• Carboysothermus hydrenoformans (RT-PCR)
• P. kodakaraensis (Thermococcus) (rapid synthesis)
• Pfu: Pyrococcus furiosus (fidelity)
– Fused to DNA binding protein for processivity
A Standard PCR Reaction Mix
0.25 mM each primer0.2 mM each dATP, dCTP,
dGTP, dTTP50 mM KCl10 mM Tris, pH 8.41.5 mM MgCl2
2.5 units polymerase102 - 105 copies of template50 ml reaction volume
Denature (heat to 95oC)
Lower temperature to 56oC Anneal with primers
Increase temperature to 72oC DNA polymerase +
dNTPs
Combinations Of Cycle Temperatures
TEMP FOR COMMENTS94-60-72 Perfect, long
primersHigher temp can be used;maximum annealling temp
94-55-72 Good or perfectlymatched primersbetween 19-24 nt
Standard conditions
94-50-72 Adequate primers Allows 1-3 mismatches/20 nt
94-48-68 Poorly matchedprimers
Allows 4-5 mismatches/20 nt
94-45-65 Unknown match,likely poor
Primers of questionablequality, long-shot PCR
94-37-65 Hail Mary Uncontrolled results
Do Errors Matter in PCR?
• Yes, if you want to clone the amplified DNA — an individual molecule may harbour several mutations.
• Use a proof-reading thermo-stable enzyme rather than Taq.
How Big A Target?
• Amplification products are typically in the size range 100-1500 bp.
• Longer targets are amplifiable — >25 kb.
• Requires modified reaction buffer, cocktails of polymerases, and longer extension times.
• Limited by the integrity of the starting target DNA — > 50 kb.
Can I PCR Amplify RNA?
• Not directly — the DNA polymerase requires a DNA template and will not copy RNA.
• mRNA can first be copied into cDNA using reverse transcriptase.
• cDNA is a template for PCR — it need not be double-stranded.
Designing PCR Primers
• Primers should be ~20 bases long.• The G/C content should be 45–55%.• The annealing temperatures should be
within 1°C of one another.• The 3´-most base should be a G or C.• The primers must not base pair with each
other or with themselves or form hairpins.• Primers must avoid repetitive DNA
Primers That Form Hairpins
• A primer may be self-complementary and be able to fold into a hairpin:
5´-GTTGACTTGATA
||||| T
3´-GAACTCT
• The 3´ end of the primer is base-paired, preventing it annealing to the target DNA.
Primers That Form Dimers
• A primer may form a dimer with itself or with the other primer.
5´-ACCGGTAGCCACGAATTCGT-3´
||||||||||
3´-TGCTTAAGCACCGATGGCCA-5´
• Primer dimers can be an excellent, but unwanted, substrate for the Taq polymerase.
Will Other Genes Amplify Too?
• The primers have been designed on the basis of the DNA sequence of a single gene.
• Might the primers also amplify other segments whose sequence we have not taken into account?
Type of PCR• Single PCR• Nested PCR• Multiplex PCR• Reverse transcriptase PCR• Asymmetric PCR• Quantitative PCR
Applications of PCRApplications of PCR
Molecular Identification Sequencing Genetic Engineering
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
• Bioinformatics
• Genomic cloning
• Human Genome
Project
• Site-directed
mutagenesis
• Gene expression
studies
SSEQUENCINGEQUENCING
Nucleotides (dNTP) are modified
(dideoxynucleotides = ddNTP)
NO polymerisation after a dideoxynucleotide!
