The exponential nature of PCR allows spectacular increases in the abun-dance of a DNA sequence being amplified. Consider a 10-kbp DNAsequence in a genome of 1010 base pairs. What fraction of the genome is rep-resented by this sequence? That is, what is the fractional abundance of thissequence in this genome? Calculate the fractional abundance of this targetsequence after 10, 15, and 20 cycles of PCR, starting with DNA represent-ing the whole genome and assuming that no other sequences in the genomeundergo amplification in the process.

PCR (polymerase chain reaction is a technique through which a DNA segment is copied to a million…

Answered: The exponential nature of PCR allows…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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The exponential nature of PCR allows spectacular increases in the abundanceof a DNA sequence being amplified. Consider a 10-kbp DNA sequence in agenome of 1010 base pairs. What fraction of the genome does this sequence represent? That is, what is the fractional abundance of this sequence in this genome?Calculate the fractional abundance of this target sequence after 10, 15, and 20 cycles of PCR, starting with DNA representing the whole genome and assuming that no other sequences in the genome undergo amplification in the process.
In the very first round of PCR using genomic DNA, the DNA primers prime synthesis that terminates only when the cycle ends (or when a random end of DNA is encountered). Yet, by the end of 20 to 30 cycles - a typical amplification - the only visible product is defined precisely by the ends of the DNA primers. Explain how.
To generate large amounts of DNA to manufacture gene therapy payloads or to be able to see them on gel electrophoresis, specific sequences can be amplified by PCR. For a sequence of a 100 base pairs, calculate the number cycles of PCR required to generate 1 ng of DNA, when starting from a single copy. Note that the molar mass of an average DNA bp is 600 g/mol.
An optimum ligation reaction should contain approximately 50 ng of vector. Given yourconcentrations recorded below, what is the volume of vector that should be added to eachligation reaction to have this mass of DNA in the reaction? Size of asPink-promoterless in bp: 702 (vector) Size of pCusC in bp: 157 The concentration of digested & purified PCR insert: 13.849 The concentration of digested and purified plasmid: 6.887 Any help with this is appreciated. I'm a bit confused thx
What is expected theoretical number of copies of DNA molecules after 28 cycles in a PCR experiment? What is the percent efficiency of the PCR experiment if the actual number of copies was 500,000,000? Show your calculations
A successful PCR experiment often depends on designing the correct primers. In particular, the T m for each primer should be approximately the same. What is the basis of this requirement?
Describe the possible outcome of a PCR experiment in which (a) there is a single-stranded break in the target DNA sequence, which is present in only one copy in the starting sample, and (b) there is a doublestranded break in the target DNA sequence, which is present in only one copy in the starting sample.
Here you see the gel electrophoresis of PCR reactions of the pMCT118 VNTR locus of 4 individuals (lanes 1-4). The outermost lanes show a DNA size standard (100bp ladder), with the fastest migrating band equal to 100bp and each successive band 100bp larger (200, 300, 400, 500, 600, etc.) Which is the smallest band of the DNA size standard? Approximately what size are all of the bands seen in lanes 1-4? Which of these four persons is homozygous for the VNTR locus? Which two individuals cannot be discriminated by this PCR?
Explain why DNA ladders are usually included during gel electrophoresis. One aspect of PCR that can be modified is the annealing temperature. In general, higher annealing temperatures show more specificity towards a single template, whereas lower annealing temperatures show less specificity and may bind to multiple regions throughout the genome. Discuss how using an annealing temperature that is too high or too low might influence the results of a PCR assay (and gel electrophoresis results) such as the one used in this study.
The final amount of each primer required in a PCR reaction is 25 picomol. If the total volume of the PCR reaction is equal to 100 µl and the stock concentration of each primer is equal to 0.0025 mM. Calculate the volume of stock primer that needs to be added in order to ensure a primer amount of 25 picomol.
The plasmid cloning vector pBR322 is cleaved with the restriction endonuclease PstI. An isolated DNA fragment from a eukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are selected by growth in the presence of tetracycline. The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one end. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below. What does each pattern say about the cloned DNA? Note: pBR322, the PstI and EcoRI restriction sites are about 750 bp apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted.
Polymerase Chain Reaction (PCR) was invented by Kary Mullis in 1983. This technique had indeed facilitated research in various areas of molecular biology and genetics. You would like to amplify a particular gene fragment from the yeast genome using Polymerase Chain Reaction (PCR). What are the THREE (3) main cycles in PCR? Discuss the processes at each PCR cycle mentioned.

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