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Lecture 36: Cloning
and Sequencing
Genes
Lecture Outline, 12/5/05
• Case Study: BRCA1, continued
– Cloning DNA fragments into plasmids
• other vectors
• “Libraries” of DNA
– Di-deoxy Sequencing
– Polymerase chain reaction (PCR)
Finding the Cancer Gene
BRCA1
• 1980’s: found several families that were
predisposed to breast cancer
• Studied 23 breast cancer families
– Early onset
– Frequent bilateral disease
– Male relatives with breast cancer
• 1990: linked the disease to a marker on
Chromosome 17q21
– D17S74 - 183rd marker used!
– Initial candidate region spanned half the
chromosome (hundreds of possible genes . . .)
Find markers that co-segregate with
the disease
1,8
2,8
1
2
4
8
4,8
2,4
1,2
Restriction enzymes cut DNA
at specific sites
*
Allele “A”
Disease
DNA probe
Allele “B”
Normal
DNA probe
AA
AB
BB
Different sequences
will have different
length fragments
BRCA1 is in the middle of
Chromosome 17--What next?
Test more
families
Try more
markers
Identify
recombinants
Recombination
Occasionally there is
a crossover during
meiosis
Marker 1
Marker 2
Marker 3
This individual
shows that the
disease must not
be near Marker 3
Why?
1
2
1
8
6
4
2
4
3
2
4
6
8
6
4
4
5
3
To find those rare
crossovers, they
needed many families
with inherited breast
cancer
Mapping BRCA1
• Larger study
• 214 breast cancer families
Chromosome 17
– Location narrowed to 8 cM
• But that was still a 600,000 nucleotide region!
• Step 2: Positional cloning to find the
actual gene
–
–
–
–
Make a “library” of cloned fragments
Order those fragments
Find fragments that contain coding sequences
Sequence those fragments
Using a restriction enzyme and DNA
ligase to make recombinant DNA
Restriction site
DNA 5
with 3
1
3
5
GAATTC
CTTAAG
Cut DNA
Restriction
enzyme, leaving
overhanging ends
G
G
Sticky end
2
Base pairing of sticky
ends produces various
combinations.
G AATT C
C TTAA G
3
DNA ligase
seals the strands.
Figure 20.3
G
G AATTC
CTTAA G
One possible combination
Recombinant DNA molecule
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
Note: The BRCA1
study used YACs
instead of
plasmids, but the
principles are
similar.
Transform
the
recombinant
plasmid into
E. coli
To produce a “library” of different DNA fragments
One of the clones in the library
should contain the gene, but
which one?
1. Probe a large insert
library to identify a
clone containing the
marker linked to the
trait.
1b. Sequence
the ends of that
fragment.
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
Chromosome walking
2. Probe again to
identify clones
containing the end
sequence of the first
clone
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
Chromosome walking
3 These clones must overlap the
first clone. Hopefully they also
contain some non-overlapping
new DNA
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
Chromosome walking
4 Again, probe the large insert library
to identify clones containing the
sequence of the ends of these clones.
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
Chromosome walking
4 Again, these clones must overlap the
existing clones. ie they have some of the
same DNA - and hopefully also some
new sequence
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
Chromosome walking
In this way we build up a CONTIG - a
series of overlapping clones centred on
our region of interest.
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
• 8 cm may have many genes, but also
lots of non-coding DNA
• Kinds of DNA sequences:
– Coding, SSR, pseudogenes, transposons
– Limit sequencing only to coding sequences
– All coding sequences make mRNA
Find the clones that contain
coding sequences
• Make a DNA copy
(“cDNA”) of the
mRNA using
Reverse
Transcriptase
• Use that to probe
for clones that
contain coding
sequences
Determining the Nucleotide
Sequence
Ingredients to synthesize DNA in vitro:
– Template DNA
– DNA polymerase
– A, C, G, T nucleotide triphosphates
– Buffer (incl. salts and MgCl)
Then “poison” this
+ One more critical ingredient:
Primer with 3’ OH
recipe with small
amounts of dideoxy
nucleotides to stop
the reaction
Di-deoxy sequencing
Precisely where the
reaction stops each
time is random, but
if there are a million
new strands
synthesized, each
possible length of
fragment will be
produced
Part of a DNA sequence
BRCA1 found in 1994
Science. 1994 Oct 7;266(5182):66-71.
A strong candidate for the breast and ovarian cancer
susceptibility gene BRCA1.
Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K,
Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al.
Department of Medical Informatics, University of Utah Medical
Center, Salt Lake City 84132.
A strong candidate for the 17q-linked BRCA1 gene, which influences
susceptibility to breast and ovarian cancer, has been identified by
positional cloning methods. Probable predisposing mutations have
been detected in five of eight kindreds presumed to segregate BRCA1
susceptibility alleles.
Results of sequencing
Many different
mutations in
BRCA1 can lead
to cancer
Now that the sequence is known, it is possible to
amplify that region from other individuals, using
PCR (polymerase chain reaction).
Overview of
PCR
Overview of
PCR
Overview of
PCR