Gene amplification through PCR Presented By P.UMA DEVI RVM 08-26 MVSC 1 ND YEAR
Contents Components of the reaction mixture PCR primer design guidelines Variations on the basic PCR technique
Comparison of PCR and Gene cloning
What is PCR?
P olymerase C hain R eaction is an in vitro technique for the amplification of a specific sequence of DNA Which is used for further testing.
Cetus Corporation (A Biotech Company of United States)
Components of the reaction mixture
Primers (forward and reverse)
Template DNA It contains the DNA region to be amplified
Range - 1-2 µl ( for a total reaction mixture of 10 µl)
Primers Short Single stranded oligonucleotides They are complementary to the 5' or 3' ends of the DNA region TT AA C GG CC TT AA . . . TTT AAA CC GG TT AA TT G CC GG AA TT . . . . . . . . . .> and <. . . . . . . . . . AAA TTT GG CC AA TT AA C GG CC TT AA . . . TTT AAA CC GG TT
Range - 1 µl ( for a total reaction mixture of 10 µl)
PCR Primer Design Guidelines Optimal length of PCR primers is 18-22 bp TT AA C GG CC TT AA ….. TTT AAA CC GG TT
AA TT G CC GG AA TT ........>
Primer Melting Temperature: (Tm) Temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability.
( GCAT no. of respective nucleotides in the primer)
Presence of G or C bases within the last five bases from the 3' end of primers
Promotes specific binding at the 3' end due to the stronger bonding of G and C bases
Primer Secondary Structures : produced by intermolecular or intramolecular interactions
Lead to poor or no yield of the product.
Hairpins Intramolecular interaction within the primer
They formed by intermolecular interactions between the two primers, where the primer is homologous to itself.
They reduce the product yield.
Repeats A di-nucleotide occurring many times consecutively. They should be avoided because they can misprime. Ex. A T A T A T A T A T A T ………..
Acceptable di-nucleotide repeats are maximum 4
Primers with long runs of a single base . Ex . A GCGGGGG A T GGGG ………..
The maximum number of runs accepted are 4
Avoid Cross homology Primers designed for a sequence must not amplify other genes in the mixture.
Position Sequence close to the 3‘ end preferred most frequently.
dNTPs De oxy nucleotide triphosphate (dATP, dGTP, dTTP, dCTP) They are the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Range - 0.5 µl (for 10µl reaction mixture)
dNTPs in the reaction mix
Taq DNA Polymerase Range 0.2ul of (in 10µl of reaction mix)
It assebles a new DNA strand from dNTPs
Contains Divalent cations like Mg+2 Provides suitable chemical environment for optimum activity and stability of the DNA polymerase Range - 1 µl ( for a total reaction mixture of 10 µl)
It’s quantity is variable
Steps in PCR
Heating the reaction to a temperature of
94-98°C for 20-30 seconds .
Denaturation of DNA template by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA .
50-65°C for 20-40 seconds Stable DNA-DNA hydrogen bonds are formed
The polymerase binds to the primer-template hybrid and begins DNA synthesis.
At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template by adding dNTPs in 5' to 3' direction.
To ensure that any remaining single-stranded DNA is fully extended. 4-15°C for an indefinite time
short-term storage of the reaction
Allele- Specific PCR Selective PCR amplification of the alleles to detect single nucleotide polymorphism (SNP)
Selective amplification is usually achieved by designing a primer such that the primer will match or mismatch one of the alleles at the 3’ end of the primer.
Asymmetric PCR It is used for DNA sequencing
The two primers are used in the 100:1 ratio so that after 20-25 cycles of amplification one primer is exhausted thus single stranded DNA is produced in the next 5-10 cycles
Real Time PCR Quantitative real time PCR (Q-RT PCR) It is used to amplify and simultaneously quantify a target target DNA molecule Real time PCR using DNA dyes
Fluorescent reporter probe method
Real Time PCR
Helicase-dependent amplification Constant temperature is used rather than cycling through denaturation and annealing/extension cycles.
DNA Helicase , an enzyme that unwinds DNA, is used in place of thermal denaturation.
Intersequence-specific PCR (ISSR):
A PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.
Inverse PCR A method used to allow PCR when only one internal sequence is known.
This is especially useful in identifying flanking sequences of various genomic inserts.
When sequence of only one end of the desired segment of gene is known,the primer complimentary to the 3' strand of this end is used to produce several copies of only one strand of the gene.
RT-PCR ( Reverse Transcription PCR) It is used to amplify, isolate or identify a known sequence from a cellular or tissue RNA . RT-PCR is widely used in expression profiling , to determine the expression of a gene or to identify the sequence of an RNA transcript.
Used to obtain 3' and 5' end sequence of cDNA transcripts
Comparison PCR - Polymerase Chain Reaction and Gene Cloning Two to four days Four hours Time for a typical experiment 12. Required Not required User’s skill 11. More Less Cost 10. Less More Applications 9. More Less Error probability 8. Yes No Labour intensive 7. No Yes Automation 6. Restriction enzymes, Ligase, vector. bacteria DNA polymerase (Taq polymerase) Biological reagents required 5. Microgram (m) Nanogram (ng) Quantity of starting material 4. Last step First step Selectivity of the specific segment from complex DNA 3. In vitro and in vivo In vitro Manipulation 2. Selective amplification of specific sequence Selective amplification of specific sequence Final result 1. Gene cloning PCR Parameter
Application of PCR Cloning a Gene encoding a known protein Amplifying cloned DNA from Vectors Rapid Amplification of cDNA ends Detecting Bacterial or Viral Infection
● Tuberculosis (Mycobacterium tuberculosis)
Diagnosing inherited disorders
Problems with PCR Polymerase lacks exonuclease activity
PCR works readily with DNA of lengths two to three thousand basepairs