Question

help with the whole question 9 please.

9. a) You are tasked with amplifying a fragment of interest from a sample of genomic DNA by using PCR List the components that need to be included in any standard PCR, give the function of each. A PCR is typically performed in an automated thermo cycling machine. Give the three steps required in each cycle, and explain the function of each. The figure below represents a step during the first PCR cycle. Indicate the polarities of both DNA strands and the primers. Use arrows to indicate the direction of synthesis of the new strands. c) Primer Primer d) You want to PCR amplify only the DNA region indicated in the block. Design a suitable forward and reverse primer each 12 bp in length, give the primers sequences 5'-3'. 5...GCCAGCCACGATATCATCGGAAGTC.............CCAGCATGCACGAGTATCGTTAAC... 3 3....CGGTCGCTGCTATAGTAGCCTTCAG.................GGTCGTACGTGCTCATAGCAATTG... 5' e) How many new dsDNA molecules will there be in a sample after five PCR cycles have been completed? Assume you started with a single copy of the target dsDNA.

Answer 1

a) Components needed for standard PCR:

1) dNTPs

2) Primers

3) Taq Polymerase

4) Buffers

5) MgCl2 (If buffers does not have)

6) Template DNA

B) Three steps of PCR reaction

1) Denaturation of template DNA : In this template DNA is denatured at high degree of temparture such as on 95 degree celcius.

2) Primer annealing: In this step primer binds to the template DNA at specific site by making complementary base pairing. The temprature of annealing depends on primer Tm.

3) Elongation: Adding nucleotides in the primers at 3' by DNA polymerase. The elongation temprature depends on the optimum temperature of DNA polymerase. For Taq polymerase optimum temperature is 72 degree celcius.

c) DNA polymerize always from 5' to 3' so labelled structure will be:

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d) F.P- 5'-ACGATATCATCG-3'

R.P- 5'-ATACTCGTGCAT-3'

e) Number of DNA strand after PCR = N * 2n

N= Initial number of dsDNA

n= number of PCR cycles

Number of DNA strand after 5 PCR cycle = 1 * 25 = 32 ds DNA.