Construction of a General Human Chromosome Jumping Library, with Application to Cystic Fibrosis

Abstract

In many genetic disorders, the responsible gene and its protein product are unknown. The technique known as "reverse genetics," in which chromosomal map positions and genetically linked DNA markers are used to identify and clone such genes, is complicated by the fact that the molecular distances from the closest DNA markers to the gene itself are often too large to traverse by standard cloning techniques. To address this situation, a general human chromosome jumping library was constructed that allows the cloning of DNA sequences approximately 100 kilobases away from any starting point in genomic DNA. As an illustration of its usefulness, this library was searched for a jumping clone, starting at the met oncogene, which is a marker tightly linked to the cystic fibrosis gene that is located on human chromosome 7. Mapping of the new genomic fragment by pulsed field gel electrophoresis confirmed that it resides on chromosome 7 within 240 kilobases downstream of the met gene. The use of chromosome jumping should now be applicable to any genetic locus for which a closely linked DNA marker is available.

An initial map of insertion and deletion (INDEL) variation in the human genome

“…One of the most common genetic diseases in humans, cystic fibrosis, is frequently caused by an allele of the CFTR gene that contains a three-base pair deletion. This deletion leads to the elimination of a single amino acid from the encoded protein, which, in turn, leads to the disease (Collins et al 1987). Trinucleotide expansion diseases, such as Fragile X Syndrome, likewise are caused by DNA insertions that result from the expansion of short trinucleotide repeat units (Warren et al 1987).…”

Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing

“…For each population/ phenotype, gDNA from each pool were then combined in equal quantity in order to be representative of a total of 130 individuals. Quantitative PCR validation of gene amplifications and genotyping of kdr mutations was performed on gDNA extracted from 12 single adult females per population/phenotype using the method described in Collins et al (1987) and resuspended in 20 µL nuclease-free water. Gene expression analyses were performed on RNA extracted from three pools of 30 adult females per population (R and S populations) using the RNAqueous4PCR kit (Life Technologies) according to the manufacturer's instructions and resuspended in 50 µL nuclease-free water.…”

Human genetics: One gene, twenty years

“…A few years previously, Collins had described the technique of 'chromosome jumping' , a way of leaping across the vast genetic distances from one marker sequence in a region to another that was much faster than the conventional way of chromosome 'walking' 10,11 . They agreed to collaborate: Collins's lab would bound to new positions, and Tsui's would walk forwards and backwards from the landing points looking for the gene.…”