Biophys Techniques: Southern Blotting, PCR, RT-PCR

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In summary, the conversation discusses different techniques used in molecular biology, including PCR, Southern blotting, reverse transcriptase PCR, and Western blotting. It is determined that Western blotting is not suitable for detecting DNA or RNA transcript, while Southern blotting can determine copy number of an insert in the animal genome. PCR can detect the presence of a gene, but not the copy number. The conversation then moves on to discussing how reverse transcriptase PCR (RT-PCR) is used to determine tissue-specific transcription levels. The substrate for RT-PCR is mRNA and the product is cDNA. However, in order to understand expression levels, a control is needed, and RT-PCR can be combined with qPCR for quantitative results.
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TytoAlba95
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Homework Statement
During transgenesis, the location of genes and their number integrated into the genome of the transgenic animal are random. It is often necessary to determine the copy number of genes and their tissue specific transcription. The following are the possible methods used for the determination:
a. PCR
b. Southern blotting
c. Reverse transcriptase PCR
d.Western blotting
Relevant Equations
Ans: Southern blotting and RT-PCR
I have problem understanding these techniques with reference to the question:
a. PCR
b. Southern blotting
c. Reverse transcriptase PCR
d.Western blotting

My attempt:
d. Western blotting is not the right method here because it doesn't detect DNA or RNA transcript.
b. Southern blotting can be used to determine the copy number of the insert in the animal genome.
a. PCR can detect the presence of the gene but cannot be used to detect the copy number.

I don't understand how RT-PCR detects the tissue specific transcription level.
 
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Hint: what are the substrate and product of reverse transcriptase?
 
  • #3
TeethWhitener said:
Hint: what are the substrate and product of reverse transcriptase?

The substrate is mRNA and product is cDNA.

Could you please explain briefly how the expression level is determined by using RT-PCR?

My understanding:
RT-PCR will simply convert all types of RNAs into cDNAs (using a oligo-dT primer) then (I don't know how the single stranded cDNA is converted into dsDNA) using a set of two gene specific primers the ds-cDNA will be amplified.

But to understand the expression level I think one needs to have a control too(if so, can you please elaborate on the control).
 
  • #4
SanjuktaGhosh said:
The substrate is mRNA and product is cDNA.

Could you please explain briefly how the expression level is determined by using RT-PCR?

My understanding:
RT-PCR will simply convert all types of RNAs into cDNAs (using a oligo-dT primer) then (I don't know how the single stranded cDNA is converted into dsDNA) using a set of two gene specific primers the ds-cDNA will be amplified.

But to understand the expression level I think one needs to have a control too(if so, can you please elaborate on the control).
Yes, you’re right. Simply using RT-PCR without any quantitation will give you a binary yes/no for whether a gene is being transcribed, but won’t give you the expression level. However, you can combine RT with qPCR to get quantitative results. Here’s an overview:
https://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/
 
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This link also provides a clear overview on qPCR.
 

1. What is Southern blotting and how is it used in biophysics?

Southern blotting is a laboratory technique used to detect and analyze specific DNA sequences. It involves separating DNA fragments using gel electrophoresis, transferring them to a membrane, and then using a labeled DNA probe to identify the target sequence. This technique is commonly used in biophysics to study gene expression, DNA methylation, and genetic mutations.

2. What is the difference between PCR and RT-PCR?

PCR (polymerase chain reaction) is a technique used to amplify a specific DNA sequence, creating multiple copies of it. RT-PCR (reverse transcription PCR) is a variation of PCR that involves converting RNA into DNA before amplification. RT-PCR is commonly used to study gene expression and detect RNA viruses.

3. How is PCR used in biophysics research?

PCR is a powerful tool in biophysics research as it allows for the amplification of specific DNA sequences, which can then be further analyzed and studied. It is commonly used in DNA cloning, genetic testing, and studying gene expression and mutations.

4. What are the advantages of RT-PCR over traditional PCR?

RT-PCR has several advantages over traditional PCR, including its ability to amplify RNA, which cannot be directly amplified by traditional PCR. It also allows for the detection of gene expression levels and the identification of RNA viruses. Additionally, RT-PCR is a more sensitive technique, making it useful for studying low levels of gene expression.

5. What are the limitations of Southern blotting?

One limitation of Southern blotting is that it is a time-consuming and labor-intensive technique. It also requires a relatively large amount of DNA for analysis, making it less suitable for studying small DNA samples. Additionally, Southern blotting can only detect specific DNA sequences and cannot provide information on the overall structure or function of DNA.

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