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Gene amplification through PCR Presented By P.UMA DEVI RVM 08-26 MVSC 1 ND YEAR

Contents PCR Definition Components of the reaction mixture PCR primer design guidelines Steps in PCR Variations on the basic PCR technique Comparison of PCR and Gene cloning Applications of PCR Problems related to PCR

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.

Kary Mullis (1987) Cetus Corporation (A Biotech Company of United States) Nobel Prize 1993

Components of the reaction mixture Template DNA. Primers (forward and reverse) dNTPs Taq DNA Polymerase Buffer solution Divalent cations Sterile deionized water

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 Range - 1 µl ( for a total reaction mixture of 10 µl) 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

PCR Primer Design Guidelines Primer Length: 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. Range - 52-58 ° C Formula Tm = 4 (G+C) + 2 (A+T) ( GCAT no. of respective nucleotides in the primer)

GC Content 40-60%. GC Clamp 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. Ex - Hairpins Dimers

Hairpins Intramolecular interaction within the primer

Self Dimer 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

Runs 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)

Taq DNA Polymerase T hermus aq aticus Range 0.2ul of (in 10µl of reaction mix) It assebles a new DNA strand from dNTPs

Buffer solution 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) Sterile deionized water It’s quantity is variable

Steps in PCR Initialization Denaturation Annealing Extension / Elongation Final elongation Final hold Initialization step Heating the reaction to a temperature of 94-96°C for 1-9 minutes.

Denaturation step 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 .

Annealing step 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.

Extension/elongation step 75-80°C At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template by adding dNTPs in 5' to 3' direction.

Final elongation 70-74°C for 5-15 minutes To ensure that any remaining single-stranded DNA is fully extended. Final hold 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

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.

Anchored PCR 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. RACE-PCR  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 Amplification of old DNA Amplifying cloned DNA from Vectors Rapid Amplification of cDNA ends Detecting Bacterial or Viral Infection ● AIDS infection ● Tuberculosis (Mycobacterium tuberculosis)

Genetics Diagnosis Diagnosing inherited disorders Cystic fibrosis Muscular dystrophy Haemophilia A and B Sickle cell anaemia Diagnosing cancer Blood group typing.

Problems with PCR Polymerase errors Polymerase lacks exonuclease activity Size limitations PCR works readily with DNA of lengths two to three thousand basepairs Non specific priming

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