What are the limitations of using an agarose gel to determine if you are homozygous or...

What are the limitations of using an agarose gel to determine if you are homozygous or heterozygous with respect to the D1S80 locus?

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Expert Solution

Homozygous : A gene having identical alleles

Heterozygous : A gene having different alleles

D1S80 locus : This locus is found on the petite arm of human chromosome consists of 16 base pairs repeat which are inherited in Mendelian fashion.

Agarose gel electrophoresis : used to determine the sizes of PCR amplified DNA fragments

The main limitations are :

1) we need to prepare 2% agarose gel which solidify very quickly than the other gels like polyacrylamide gel because of high concentration of agarose. So we need to melt when required in a microoven.

2) As the percentage of gel is high it is brittle and may not set uniformly.

3) Agarose gels will not have a uniform pore size.

4) During interpretation we can observe a single band which indicates homozygous at D1S80 locus.

But there is a possibility that two different alleles of nearly equal size can also been intrepreted as homozygous because agarose gel electrophoresis cannot resolve them.

4) If two bands are present it is indicated as heterozygous at D1S80 locus but larger alleles are efficiently amplified than the smaller ones.

5) Need to take care in the preparation of agarose gel this may lead to no bands.

gladiator answered 1 year ago

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