Basic PCR Technique. Presented by : Noorul Hidayah Badri

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1 Basic PCR Technique Presented by : Noorul Hidayah Badri

2 What is PCR? PCR is aninvitro technique which allow the amplification of a specific DNA region. PCR is like selecting a specific page from book and photocopying it millions of times. PCR is one of the most useful techniques in laboratories due to its speed and sensitivity.

3 PCR allows exponential amplification of DNA sequence - each PCR cycle theoretically doubles the amount of DNA - during PCR, an existing DNA molecule is used as a template to synthesize a new DNA strand. - through repeated cycles of DNA amplification, large quantities of DNA are produced.

4 Figure 1: PCR cycle

5 Buffer MgCl 2 Primer PCR components dntp DNA DNA Polymerase Enhancer

6 How does PCR amplify DNA? Three step: Denaturation separate the strands of the double stranded cellular DNA Annealing Lower the temperature to allow primer bind to their complementary sequences Extension polymerase synthesis DNA strands using the 3 end of the primer as the start point and the cellular DNA strand as template.

7 Parameter for optimization of PCR Many parameters need to be optimized to increase the yield and sensitivity of detection or amplification: These parameter include: - PCR thermal profile (primer annealing, extension, denaturation, number of cycles ) - MgCl 2 concentration - Primer design and concentration -Template quality and quantity

8 PCR thermal profile optimal annealing temperature depends on the length and composition of nucleotides PCR primers. Number of cycle usually between cycle number efficiency of the amplification decreased as primer & nucleotides are consumed and polymerase start to loose it activity. no increase in the desired product

9 MgCl 2 concentration Generally insufficient MgCl 2 leads to low yield and excessive MgCl 2 will result in the accumulation of nonspecific products. Optimal MgCl 2 concentration depend on the primers, DNA polymerase, template, and dntps concentration.

10 Primer design and concentration The optimum length of a primer is generally from 20 to 30 nucleotides with a melting temperature between 55 C and 65 C. If possible design the forward and reverse primer with same melting temperatures and GC content. Sequences possessing significant secondary structure should be avoided.

11 DNA quality and quantity DNAshouldbeintactandfreefrominhibitorthatcan inhibit PCR amplification. - inhibitor can be from original DNA sources ( heme from blood, melanin from hair) - inhibitor can be introduced during DNA extraction process ( phenol, sodium dodecyl sulfate (SDS), ethanol and others.) More template is not necessary better - too much DNA can cause nonspecific amplification. - too little DNA will result in little or no amplification.

12 PCR animation

13 References C.R. Newton & A. Graham (1997) PCR. BIOS Scientific Publishers. Daniel J. F. &AndersenW. R. (1999) Genetic. Wadsworth Publishing Company

14 Thank you

15 Buffer Chemical solution that provides the optimal environmental condition. 10X buffer mm KCl mmtris HCl - 15mM MgCl 2

16 MgCl 2 a necessarycofactor for DNA polymerase activity Cofactor - An organic molecule or ion (usually a metal ion) that is required by an enzyme for its activity.

17 Primer a strand of nucleic acid that serves as a starting point for DNA amplification.

18 Deoxynucleoside Triphosphate (dntps) the building blocks from which the DNA polymerase synthesizes a new DNA strand. - deoxy adenosine triphosphate (datp) - deoxy cytidine triphosphate (dctp) - deoxy guanosine triphosphate (dgtp) - deoxy thymidine triphosphate (dttp)

19 DNA Serve as template for replication

20 DNA polymerase Catalyze the synthesis of long polynucleotide chain from monomer deoxynucleoside triphosphate using one of the original parental strands as a template for the synthesis of new complementary strands.

21 Enhancers/Additives A variety of PCR additives and enhancing agents used to increase the yield, specificity and consistency of PCR reactions.

22 Table 1: Enhancer of PCR Substance Formamide 5% dimethyl sulfoxide (DMSO) < 10% Concentration Tetramethylammonium chloride (TMAC) µm Polyethylene glycol 600 (PEG) 5-15% Glycerol 10-15% Tween % Gene 32 protein 1 nm 7 deaza-dgtp Replace 75 % of dgtp with deaze-dgtp

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