PCR Amplifies Targeted Sequence

Transcription

1 PCR Amplifies Targeted Sequence Target Sequence Supercoiled DNA Strand DNA Strand Double Helix DNA Strand Chromosome

2 P C R

3 PCR PCR = Polymerase Chain Reaction. A primer directed-extension reaction for amplifying specific DNA sequence in vitro, using heat-stable DNA polymerase (Taq DNA polymerase). PCR involves repetitive (>30) or cycles of: Denaturation (melting of DNA to expose base composition), Annealing of primers: defining region to be amplified Polymerization (to synthesize DNA). The purpose of PCR is to make a large number of copies of a gene or gene segments.

4 The Nucleotide Sequence Hydrogen Bonds Cytosine (C) Adenine (A) Thymine (T) Guanine (G) Guanine (G) Thymine (T) Adenine (A) Cytosine (C) Deoxyribose (Sugar molecule) Phosphoric Acid (Phosphate molecule)

5 PCR STEPS Initial Denaturation: Performed only once at the start of PCR Usually at 95 C for 3-5 min. Ensures melting of genomic double-stranded DNA. Cycling Reactions Denaturation usually at 94 C for sec double strand melts (opens) to single stranded DNA. All enzymatic reactions stop. Annealing Most variable step: C for sec Primers that fit exactly on double-stranded template DNA bind tighter. Extension Taq DNA polymerase optimal temperature: C. Taq DNA polymerase adds dntp in the 5' 3' direction. Final Extension Varies from 5-10 min. All single-stranded DNA is double-stranded.

6 PCR Cycle - Step 1 Denaturation by Heat Target Sequence Target Sequence

7 PCR Cycle - Step 2 Biotinylated Primers Anneal to Ends of Target

8 PCR Cycle - Step 3 Taq DNA Polymerase Catalyses Primer Extension as Nucleotides are Added

9 End of the 1st PCR Cycle Two Copies of Target Sequence

10 Target Amplification No. of Cycles No. Amplicon Copies of 1 cycle = 2 Amplicon 2 cycle = 4 Amplicon 3 cycle = 8 Amplicon 4 cycle = 16 Amplicon 5 cycle = 32 Amplicon 6 cycle = 64 Amplicon 7 cycle = 128 Amplicon ,048, ,

11 Difference between RNA and DNA RNA DNA Sugar Ribose Deoxyribose Adenine (A) Adenine (A) Bases Cytosine (C) Cytosine (C) Uracil (U) Thymine (T) Guanine (G) Guanine (G) Strands Usually single Double Heat stable? No Yes

12 Reverse Transcription - Step 1 Biotinylated Primer Anneals to Target RNA

13 Reverse Transcription - Step 2 rtth DNA Polymerase Catalysing Primer Extension by Incorporating Nucleotides

14 End of Reverse Transcription - Step 3 - Synthesis of Complementary DNA (cdna) to the RNA Target Sequence

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16 ASESSING PCR SUCCESS There is a product formed. The product is of the right size. The amplified segment must have the correct size (in bp) Only one product is formed

17 OPTIMIZATION OF PCR PRIMER DESIGN Primer length Melting Temperature (Tm): Tm = 2(A+T) + 4(G+C) Specificity Complementary primer sequences: no intra-primer homology G/C content, polypyrimidine (T/C), polypurine (A/G) stretches. Taq DNA Polymerase Concentration Touch-down PCR Hot-start method

18 PCR PRIMERS Primers are complementary to sequences surrounding the amplified segment. PCR primers: bp long, complementary to one strand (5' 3') upstream and complementary to the opposite strand (5' 3') downstream. Must have no intra-primer or inter-primer homology, with balanced G-C vs. A-T content (typically 45-55% GC): Too many G or C stretches = non-specific annealing. Too many A and T stretches = premature opening of the primer-template complex. A poorly designed primer can result in a failed or nonspecific PCR, and can, and/or to formation of primer-dimer artefact. The primer sequence determines: length of the product melting temperature product yield. The target sequence to be amplified is usally bp in length.

19 Melting Temperature (Tm) Defined as the temperature at which half (50%) of the primers will form duplex with complementary primer at the same concentration. The annealing temperature is set few degrees ( 5 C) lower than the melting temperature: for annealing temperature of 50 C, the calculated melting temperature(t m ) ~55 C. Both PCR primers must have similar T m : primer mismatch at T m results in less efficient, or no PCR: Primer with the higher T m will mis-prime at lower temperatures Primer with the lower T m may not work at higher temperatures.

20 Calculating Tm Tm ( C)= 4(G + C) + 2(A + T) Example: CCA TCC CTT CCT CCA AAT AGA T 1G + 9C; 6A + 6T Tm = 4(1 + 9) + 2(6 + 6) = 64 C CTT CCA CAC CCT AGT TTA GTG ACA A Tm =.

21 Plateau Effect Utilization of substrates (dntps or primers) Stability of reactants (dntps or enzyme) End-product inhibition (pyrophosphate, duplex DNA) Competition for reactants by nonspecific products or primerdimer Reannealing of specific product. Incomplete denaturation/strand separation of product at high product concentration.

22 OPTIMIZATION OF PCR Cycling conditions: Cycle times Denaturation Annealing Extension Magnesium Ion Concentration Primer and Nucleotide Concentration Plateau Effect

23 TROUBLESHOOTING PCR All reagents should be kept on ice Top vs. bottom-heated. Aliquot reagents into smaller volumes Use aerosol-resistant tips (ART) DNA ladder (standard): the size of the PCR product MUST correspond to the fragment size Negative and positive control DNA Avoid overflow of sample from one well to another.

24 Aerosol-resistant tips (ART)

25 PCR Instruments: Old and New