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PCR fragment analysis - please help!

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PCR fragment analysis - please help!

Postby Sarah Spencer » Sun May 01, 2005 11:31 am

Hi people, I'd really appreciate a bit of help with this question:

A human disease is investigated at the DNA level using PCR followed by restriction enzyme analysis with HinDIII. The gene responsible for the disease has two alleles (A1 and A2). In both cases, the PCR generates a product of 900 base pairs; the enzyme HindIII only cleaves the PCR product of one of the two alleles to generate fragments of 500 and 400 base pairs. A couple with an affected son and a healthy daughter and expecting twins (one male and one female) have been genetically tested for this disease and the sizes of the DNA fragments obtained after PCR and restriction enzyme analysis are as follows: Father (900, 500, 400), Mother (500, 400), Affected son (900, 500, 400), Healthy daughter (500, 400), Male foetus (500, 400), Female foetus (900, 500, 400). I've been asked to (a) determine the genotype of the father, the mother, the two children and the foetuses; (b) identify the mutant allele; (c) discuss the genetic make-up of the twin foetuses and whether they are likely to be affected by the disease; (d) classify this disease (autosomal or X-linked; dominant or recessive). If you can help explain how to answer this question, I'd be very grateful.

Thanks, Sarah
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Postby GreenDog » Mon May 02, 2005 5:10 pm

This looks so familiar… and here is the answer:

The phenomenon observed in this question is SNP (Single Nucleotide Polymorphism), in which a mutation of one nucleotide causes the loss of a restriction site.
Let us define: 500/400 fragments = allele A 900 fragment = allele a.
The father is 900, 500/400 so he is heterozygous and his genotype is Aa (he is affected too). The mother is 500/400 so she is AA. The affected son is like the father (Aa) and the healthy daughter is like her mother (AA). The male twin is AA (like the mother) and the female twin is Aa and affected (like the father).
Whoever gets the 900 fragment has the mutation, and doesn’t have the restriction site. He also carries the disease. We can also see from the data (the heterozygous sick son) that a is dominant over A.
The disease is autosomal, because the father inherits it to sons and daughters (think of it).

Hope I've been helpful.
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