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Concept explainers
A1.
To explain: The number of DNA fragments labelled in gray, green, or red, or outlined in yellow is produced if the PCR shown in Fig 10-15 runs additional two rounds of amplification.
Introduction: Polymerase chain reaction (PCR) is adevice used to amplify small fragments of DNA for various analyses. PCR is a cost-effective, dependable, and a simple way to repeatedly amplify, that is,to replicate a small fragment of DNA of interest. PCR is the most widely used molecular technique all over the world. PCR is done in simple steps:Denaturation of the DNA fragmentàAnnealing of the primersàElongation of the DNA fragment along the primer sideàDenaturationàCycle continues until the required DNA quantity is obtained. PCR is exclusively quantitative.
A1.
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Explanation of Solution
The number of DNA produced in a generation is 2n, where ‘n’ is the number of generations. At the end of one additional round of amplification which is 25=32 DNA, there will be gray-2; green-4; red-4; and yellow outlined-22 fragments. At the end of one more additional round of amplification which is26=64 DNA, there will be gray-2; green-5; red-5; and yellow outlined-52 fragments. 2n-2 is the actual formula, where 2 is the parent DNA being conserved. Therefore, all generations has gray-2 in number.
A1.
To explain: The fragment that will predominate after several additional cycles.
Introduction: Polymerase chain reaction (PCR) is adevice used to amplify small fragments of DNA for various analyses. PCR is a cost-effective, dependable, and a simple way to repeatedly amplify, that is,to replicate a small fragment of DNA of interest. PCR is the most widely used molecular technique all over the world. PCR is done in simple steps:Denaturation of the DNA fragmentàAnnealing of the primersàElongation of the DNA fragment along the primer sideàDenaturationàCycle continues until the required DNA quantity is obtained. PCR is exclusively quantitative.
A1.
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Explanation of Solution
The DNA fragments highlighted in yellow will increase exponentially and in the end dominates the others after several additional cycles. DNA sequence that spans the distance between the two primers + length of the primers = Total length of the DNA sequence that predominates.
B.
To calculate: The number of cycles of PCR amplification required to produce 100ng of DNA from a double-stranded DNA with 500
Introduction: Polymerase chain reaction (PCR) is adevice used to amplify small fragments of DNA for various analyses. PCR is a cost-effective, dependable, and a simple way to repeatedly amplify, that is,to replicate a small fragment of DNA of interest. PCR is the most widely used molecular technique all over the world. PCR is done in simple steps:Denaturation of the DNA fragmentàAnnealing of the primersàElongation of the DNA fragment along the primer sideàDenaturationàCycle continues until the required DNA quantity is obtained. PCR is exclusively quantitative.
B.
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Explanation of Solution
Calculation:
1 ds DNA = 500 Nucleotides pairs=1000 Nucleotides
Average molecular mass of 1 nucleotide=330 g/mole(Required weight of DNA from 500 nucleotide pairs ,W)= 100 ng=10−7 g
Mass of 1 ds DNA, W0=Total No. of nucleotides×Average mass of 1 NucleotideAvagadro constant=1000×330 g/mole6×1023/mole=336×103+1−23=5.5×10−19g
To find the number of PCR cycles:
Final weight of DNA=Initial weight of DNA ×2Nwhere N= number of PCR cyclesTharefore, W=W0×2N10−7 =5.5×10−19×2N
10−7+19 =5.5×2N1012 =5.5×2N
Taking log10 on both sides:log 1012 =log(5.5×2N)12log10=log5.5+Nlog212=0.7+N×0.3N=12−0.70.3
=11.30.3=37.6 cycles≈40 cycles
Thus, approximately 40 cycles of PCR are required to amplify 500 nucleotide pairs double-stranded DNA to obtain 100 ng of DNA. 100 ng of DNA is the minimum amount of DNA required for biochemical analysis. As PCR is automated, if the protocols are followed correctly, the entire procedure will take less than a day.
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