5. a92737
Why gene identification was important?
It is one of the commonest severe Mendelian diseases in Europe
and the USA and one in which all attempts to identify the
underlying defects through biochemical and physiological
approaches had failed.
What were the problems faced?
• The disease was autosomal recessive and families were generally small
in the countries where it was prevalent.
• Mapping was done which relied on sib pairs and hoping that there was
no heterogeneity.
• The first linkage was found on locus encoding the enzyme paraoxonase
, whose chromosome location was unknown. This was rectified when
paraxonase and hence CF were localized to chromosome 7q31
a
6. a
US Canadain group led by Lap-chee Tsui used 12 different genomic libraries,
including one made from a human-hamster hybrid.
Chromosome jumping was used to bypass the size limitation of cosmid, which have maximum
cloning capcity of 45kb.
This was an early version of pair-end mapping technique that is now used to scan human genome
for structual variants.
7. Genomic DNA
Partial restriction
digestion
80-130 kb fragment, some
containing a known sequence at
one end and target sequence at
other end
Short marker
sequence
Circles with one or more copies of marker
sequence at junction plus concatamers of
marker sequence
• Digest into fragments of 10kb
• Clone into λ phage
• Select phage containing both
known sequence and target
sequence
Figure 2
8. a
DNA fragments of 80-130 kb from partial MboI digest of genomic DNA were recovered
from pre-parative pulsed-field gels
Fragments were mixed with short sequence that had sticky ends and then exposed to
DNA ligase.
Circular product were fragmented with restriction enzyme and then cloned
The desired clone contained the marker and known 7q31 sequence, joined to unknown
sequence, which was hoped to come from a position in 7q31 that was 80-130 kb away
from known sequence.
9. a
Restriction map was constructed across the region and the available genomic clones were
hybridized to zoo blots to find genomic sequences that were conserved across the species.
Such sequence were putative gene sequences.
Segments that showed conservation across species were used to screen cDNA libraries
made from tissues that were affected in CF.
A search was made for CpG islands that often mark 5' end of genes and clones were
sequenced to look for open reading frame.
Eventually, a 6.5 kb cDNA clone was isolated that lead to characterization of CF
transmembrane receptor (CFTR) Gene.
10. a
Learning outcomes
Cloning from DNA library was to identify the gene.
Human-Hamster hybrid cells containing only human chromosome 7 was used for
cloning without any clue for position of the candidate gene.
Chromosome jumping approach was used to get clones that contained the gene
of interest. Comparison with other species was carried out and conserved
sequence were used to screed cDNA library from affected muscle genes, with
CpG island as a marker for the 5’ end of gene. 6.5 kb fragment was found to
contain CFTR gene
11. References
Figure 1: Nicholas J. Magon, Rufus Turner, Richard B. Gearry, Mark B. Hampton, Peter D. Sly, Anthony J.
Kettle, Oxidation of calprotectin by hypochlorous acid prevents chelation of essential metal ions and
allows bacterial growth: Relevance to infections in cystic fibrosis, Free Radical Biology and Medicine,
Volume 86, 2015, Pages 133-144, ISSN 0891-5849,
https://doi.org/10.1016/j.freeradbiomed.2015.05.022.
Figure 2: Strachan T., Read A. (2010). Human molecular genetics. 4th edition. Garland science
Strachan T., Read A. (2010). Human molecular genetics. 4th edition. Garland
science
