Genotyping of ALDH2 gene by allele-specific PCR

What is the purpose of genotyping the ALDH2 gene by allele-specific PCR?

To determine the presence or absence of the SNP allele at codon 504.

What is the reaction catalyzed by aldehyde dehydrogenase?

CH3CH2OH → CH3CHO (acetaldehyde) → CH3COO- (acetate).

What is the result of deficiency in the ALDH2 gene?

Accumulation of acetaldehyde leading to alcohol flush response.

What is the SNP in the ALDH2 gene?

rs671: SNP (G to A) at codon 504.

What is the purpose of allele-specific PCR?

To determine the presence or absence of a specific SNP allele.

What are the methods for SNP genotyping?

Allele-specific PCR, PCR-RFLP, DNA sequencing, microarray, primer extension.

What is the purpose of PCR?

To amplify a specific DNA fragment from a complex mixture of DNA molecules.

What are the three steps in PCR?

Strand separation, primer annealing, strand elongation.

What is the purpose of strand separation in PCR?

To denature the template DNA.

What is the purpose of primer annealing in PCR?

To allow the primers to bind to the DNA template.

What is the purpose of strand elongation in PCR?

To synthesize new DNA strands using the primers as a starting point.

What is the general principle of allele-specific PCR?

The presence or absence of PCR product indicates the presence or absence of the target allele.

Why is DNA polymerase without 3' to 5' exonuclease activity required for allele-specific PCR?

To prevent removal of the mismatched base at the 3' end of the growing DNA strand.

What components are required for allele-specific PCR?

Buffer, template DNA, primers, dNTPs, thermostable DNA polymerase, divalent cation (Mg2+).

What factors should be considered in primer design for PCR?

Avoid primer dimers, avoid inverted repeats, 18-30 nucleotides long, appropriate annealing temperature.

What is multiplex PCR?

Amplifying more than one gene in a single PCR reaction.

What is the purpose of agarose gel electrophoresis?

Analysis and purification of DNA fragments for cloning.

How does DNA migrate in agarose gel electrophoresis?

Smaller molecules migrate faster towards the positive electrode.

What is the principle of agarose gel electrophoresis?

DNA or RNA molecules migrate according to their size.

What equipment is required for agarose gel electrophoresis?

Agarose, electrophoresis chamber and power supply, gel casting tray, sample combs, electrophoresis buffer.

What are the different types of electrophoresis buffers?

TAE and TBE.

What is the purpose of loading dye or buffer in agarose gel electrophoresis?

To provide sample and monitor the progress of electrophoresis.

What is the purpose of DNA intercalating dye in agarose gel electrophoresis?

To visualize DNA bands.

What is the optimal resolution of DNA in agarose gel electrophoresis?

Depends on the concentration of agarose.

What factors affect agarose gel electrophoresis of DNA?

DNA size, concentration of agarose, voltage, DNA topological form.