Cloning genomic DNA into EMBL3

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Cloning genomic DNA into EMBL3
1) Cleave EMBL3 with BamHI (GGATCC)
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2) Ligate 17kb Sau3A cleaved genomic DNA into the
l arms
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Non-recombinants can be reduced by cleaving the
stuffer with EcoR1
Recombinant molecules are in-vitro packaged to
give mature intact l phage particles which are
used to infect E. coli to give plaques.
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Genomic libraries were used to locate the cystic
fibrosis gene

1. Linkage analysis identified markers which
   segregated with the CF phenotype.
2. Library was constructed, and screened using the
   XV-2c marker.
3. Hybridising clones were used to rescreen the
   library to produce a contig.
4. 4 candidate genes were identified (using
   sequencing) on this contig.

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Genomic libraries were used to locate the cystic
fibrosis gene
5) Only one of the 4 genes hybridised to mRNA
from the pancreas and lungs. 6) CF gene extends
over 250 kb, and is involved in chloride
transport.
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Once an overlapping library(ies) has been
produced, individual clones are shotgun sequenced
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In shotgun sequencing, individual clones are
randomly fragmented and randomly sequenced
Random sequence is assembled together to give a
contig
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The human genome has been sequenced
using libraries constructed in YAC and BAC
vectors.
Yeast Artificial Chromosome
Bacterial Artificial Chromosome
YAC vectors can accept up to 1000 kb
human genome can be represented in 10,000 YAC
clones
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The libraries were prepared from DNA obtained
from anonymous
human donors
The opportunity to donate DNA for this purpose
was broadly advertised near the two laboratories
engaged in library construction
Volunteers of diverse backgrounds were accepted
on a first-come,
first-taken basis.
The samples were made anonymous as follows
The sampling laboratory stripped all identifiers
from the samples, applied random numeric labels,
and transferred them to the processing
laboratory, which then removed all labels and
relabelled the samples
All records of the labelling were destroyed. The
processing laboratory
chose samples at random
The identity of the donors for the libraries is
not known, even by
the donors themselves
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Samples were sequenced robotically on automated
sequencers.
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Major findings of the Human Genome Sequencing
Project
We have many fewer genes than might have been
expected for a relatively complex organism. Flies
have 13,000 genes, nematode worms have 18,000 and
thale cress Arabidopsis thaliana) has 26,000.
From analysis of the human draft genome, there
only seem to be 30,000-40,000 genes.
Remember Alternate splicing!!!!!!
More than half of the euchromatic genome is
comprised of repeat sequences, with the vast
majority (45) accounted for by repeats derived
from parasitic DNA, called 'transposable
elements' or 'transposons' which are SINES
Bacteria have also left their mark on our
genome. Remarkably, 223 genes found in human are
more similar to bacterial genes than to anything
seen in yeast, worm, fly or plants. And they
appear to have been transferred from a range of
bacterial species.