Lectures 22-24: Cloning human disease genes

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since 4 October 1999

(version 23 September, 1999)

1. By complementation:

a. Obtain a library of DNA:

b. Transfect mutant cells with a phenotype, e.g. X-ray sensitivity

c. Look for X-ray resistant cell lines

d. Do they carry the wild-type version of the gene?

2. Positional Cloning: Isolating the gene on the basis of its position in the genome.

a. Chromosome rearrangement

b. RFLPs (restriction fragment length polymorphisms) – detected by Southern blot.

Southern Blot:

RFLP analysis

The D allele is linked to RFLP #1. Which child is a recombinant?

c. Microsatellite markers – detected by PCR (polymerase chain reaction),

PCR:

link to http://www.blc.arizona.edu/courses/181gh/

Microsatellite marker analysis:

Which microsatellite allele is likely linked to the disease allele?

Note microsatellites are more common than RFLPs, easier to detect, show a higher degree of heterozygosity.

Statistical analysis of linkage data:

Use LOD (logarithm of the odds) score

LOD = log10[(the probability of obtaining the observed progeny if the two genes are linked with a recombination frequency theta)/(the probability of obtaining the observed progeny if the two genes are unlinked)]

e.g., Suppose for two genes and a recombination frequency of 0.1 map unit the LOD score is 3. Then the odds are 1000 to 1 in favor that these data resulted from the two genes being 0.1 map unit apart, rather than from a statistical fluke in the segregation of two unlinked genes.

LOD score of 1 means 90% chance linkage is real

LOD score of 2 means 99% chance linkage is real

LOD score of 3 means 99.9% chance linkage is real etc.

In practice, a lod score greater than 3 is taken to indicate linkage.