Determine whether traditional polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), or quantitative PCR(qPCR) should be used for each applicationPCRRT-PCRДРCRcloning DNA sequencesdetecting differential exon usegenotypingcloning MRNA sequencesverifying microarray dataquantification ofgene expressionAnswer Bankо

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Asked Nov 15, 2019
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I am having issues with differentiating between the different types of PCRs and why the different applications belong to those categories

Determine whether traditional polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), or quantitative PCR
(qPCR) should be used for each application
PCR
RT-PCR
ДРCR
cloning DNA sequences
detecting differential exon use
genotyping
cloning MRNA sequences
verifying microarray data
quantification of
gene expression
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Determine whether traditional polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), or quantitative PCR (qPCR) should be used for each application PCR RT-PCR ДРCR cloning DNA sequences detecting differential exon use genotyping cloning MRNA sequences verifying microarray data quantification of gene expression Answer Bank о

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Expert Answer

Step 1

The polymerase chain reaction (PCR) is a technique in which several million copies of the desired DNA sequence are rapidly produced in a test tube. Even a trace amount of DNA obtained from a hair follicle or a skin cell can give adequate genetic information to reveal a unique DNA profile of an individual.

Traditional PCR setup includes the target DNA sequence, DNA polymerase enzymes such as Taq polymerase which is thermostable, a supply of the four types of DNA nucleotides (adenine, guanine, cytosine, and thymine), artificially made primers that are complementary to the target sequence, and a thermal cycler that controls the temperature. Initialization, Denaturation, Annealing, elongation are the steps of PCR.

RT-PCR is also known as Reverse Transcriptase PCR. It is a variation of the traditional polymerase chain reaction that typically measures RNA expression levels. During the process of RT-PCR, complementary DNA or cDNA is produced by reverse transcribing of the RNA templates with the enzyme reverse transcriptase.

Quantitative PCR (qPCR) is also known as real-time PCR. It is used to detect, characterize and quantify DNA sequences for numerous applications. In qPCR, RNA transcripts are measured by copying them into cDNA initially, as described in the RT-PCR and then qPCR is subsequently carried out. In qPCR, fluorescent labeling enables the data collection by applying the use of a dsDNA binding dye. During each cycle, the fluorescence is measured. The fluorescence signal is directly proportional to the amount of replicated DNA and hence the DNA is measured in real-time.

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Step 2

Traditional PCR Applications:

Traditional PCR measures the amount of PCR product collectively at the end of each PCR cycle. The absolute quantity of products cannot be measured by using this method.

DNA cloning:

It is the process in which multiple, identical copies of a particular piece of DNA are produced. In a traditional PCR, DNA is cloned into multiple copies.

Genotyping: 

It is the process of determining differences in the genotype of species. Using the traditional PCR method DNA sequence can be analyzed and genotyping of an individual can be achieved.

Step 3

RT-PCR Applications:

Cloning mRNA sequences:

Reverse transcription PCR (RT-PCR) uses mRNA instead of DNA, as the initial template. First, the mRNA is used as a template by the enzyme “reverse transcriptase” to produce a complementary single-stranded DNA or cDNA. This process is known as reverse transcription of mRNA. Subsequently, DNA polymerase is used to convert this single-stranded cDNA into double-stranded DNA. These DNA molecules serve as templates for a PCR reaction.

Detecting differential exon use:

Exons are composed of coding sequences that carry the information for “protein synthesis.” Before the translation process, pre-mRNA introns are removed and exons are combined. This process is known as “splicing” which produces mature mRNA. Exons with the most significant ...

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