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Questions and Answers
What was the procedure devised by Alwine et al. (1978) for?
What was the procedure devised by Alwine et al. (1978) for?
- Determining the number of genes present on a given DNA
- Transferring proteins from acrylamide gels to membranes
- Transferring DNA bands from gel to chemically reactive paper
- Transferring RNA bands from gel to chemically reactive paper (correct)
What is the theoretical advantage of Dot blotting?
What is the theoretical advantage of Dot blotting?
- Transferring RNA bands from gel to chemically reactive paper
- Determining the number of genes through mRNA present on a given DNA (correct)
- Modifying Southern and Northern blotting techniques
- Transferring proteins from acrylamide gels to membranes
What are the drawbacks of Northern blotting?
What are the drawbacks of Northern blotting?
- Each gene may give rise to two or more RNA transcripts, the presence of introns
- Each gene may give rise to two or more RNA transcripts, the absence of exons and introns
- Each gene may give rise to two or more RNA transcripts, the presence of exons and introns (correct)
- Each gene may give rise to only one RNA transcript, the presence of exons and introns
Which technique is used to transfer proteins from acrylamide gels to membranes?
Which technique is used to transfer proteins from acrylamide gels to membranes?
Who coined the term 'western' blotting?
Who coined the term 'western' blotting?
What technique is a modification of Southern and Northern blotting techniques?
What technique is a modification of Southern and Northern blotting techniques?
Who is the scientist that developed the Southern blotting technique in 1975?
Who is the scientist that developed the Southern blotting technique in 1975?
What is the first nucleic acid blotting procedure developed in 1975?
What is the first nucleic acid blotting procedure developed in 1975?
In Southern blotting, which type of DNA migrates towards the anode?
In Southern blotting, which type of DNA migrates towards the anode?
What happens to smaller DNA fragments in Southern blotting?
What happens to smaller DNA fragments in Southern blotting?
After denaturation and transfer in Southern blotting, DNA fragments are transferred to which material?
After denaturation and transfer in Southern blotting, DNA fragments are transferred to which material?
What is the purpose of Southern blotting?
What is the purpose of Southern blotting?
What happens to DNA when exposed to heat (80°C) in a Southern blotting technique?
What happens to DNA when exposed to heat (80°C) in a Southern blotting technique?
What helps draw the transfer buffer through the gel in Southern blotting?
What helps draw the transfer buffer through the gel in Southern blotting?
What are the probes used in Southern blotting exposed to the nitrocellulose or nylon paper?
What are the probes used in Southern blotting exposed to the nitrocellulose or nylon paper?
What does the autoradiograph reveal in a Southern blotting technique?
What does the autoradiograph reveal in a Southern blotting technique?
What is a variant of Southern blotting used for specific analysis?
What is a variant of Southern blotting used for specific analysis?
What is the purpose of hybridization membrane in Southern blotting?
What is the purpose of hybridization membrane in Southern blotting?
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Study Notes
Blotting Techniques
- Western blotting is used to transfer proteins from acrylamide gels to membranes.
- Northern blotting is a technique for determining the number of genes (through mRNA) present on a given DNA.
- Southern blotting is a procedure in which RNA bands are blot transferred from the gel onto chemically reactive paper, where they are bound covalently.
- Dot blotting is a modification of Southern and Northern blotting techniques.
Southern Blotting Procedure
- Genomic DNA is isolated from cells/tissues and digested with one or more restriction enzymes.
- The mixture is loaded into a well in an agarose or polyacrylamide gel and then subjected to electrophoresis.
- DNA, being negatively charged, migrates towards the anode; smaller DNA fragments move faster.
- The separated DNA molecules are denatured by exposure to a mild alkali and transferred to nitrocellulose or nylon paper.
Transfer of DNA Molecules
- A nitrocellulose or nylon paper is sandwiched between the gel and a stack of paper towels, which draws the transfer buffer through the gel by capillary action.
- The DNA molecules are carried out of the gel by the buffer flow and immobilized on the membrane.
Hybridization and Autoradiography
- The nitrocellulose or nylon paper is exposed to labeled cDNA probes, which hybridize with complementary DNA molecules on the paper.
- The paper, after thorough washing, is exposed to X-ray film to develop autoradiograph, revealing specific bands corresponding to the DNA fragments recognized by the probe.
Variants of Blotting Techniques
- Eastern blotting is a variant of Southern blotting used for specific analysis.
- Dot blotting is a variant of Southern and Northern blotting techniques.
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