Genetics of Cancer

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Lecture 36 Cloning and Sequencing Genes
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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)

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Finding the Cancer Gene BRCA1

• 1980s 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 . . .)

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Find markers that co-segregate with the disease
1 , 8
2 , 4
2 , 8
4 , 8
1 , 2
1
2
4
8
5
Restriction enzymes cut DNA at specific sites

Disease
Allele A
DNA probe
Normal
Allele B
DNA probe
Different sequences will have different length
fragments
AA AB BB
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BRCA1 is in the middle of Chromosome 17--What
next?
Test more families
Try more markers
Identify recombinants
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Recombination
Occasionally there is a crossover during meiosis
1 2 1
8 6 4
Marker 1 Marker 2 Marker 3
2 4 6
4 5 3
To find those rare crossovers, they needed many
families with inherited breast cancer
This individual shows that the disease must not
be near Marker 3 Why?
2 4 3
8 6 4
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Mapping BRCA1
Chromosome 17

• Larger study
• 214 breast cancer families
• 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

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Using a restriction enzyme and DNA ligase to make
recombinant DNA
Restriction site
5?
3?
DNA
G A A T T C
Cut DNA with Restriction enzyme, leaving
overhanging ends
3?
5?
C T T A A G
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A A T T C
G
C T T A A
G
Sticky end
Fragment from differentDNA molecule cut by
thesame restriction enzyme
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A A T T C
Base pairing of sticky ends produces various
combinations.
G
G
C T T A A
G
A A T T
C
A A T T C
G
C T T A A
T T A A
G
G
C
One possible combination
DNA ligaseseals the strands.
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Figure 20.3
Recombinant DNA molecule
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Note The BRCA1 study used YACs instead of
plasmids, but the principles are similar.
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Transform the recombinant plasmid into E. coli
To produce a library of different DNA fragments
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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.uk8080/bio/studyweb/
modules/BIOL315/
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Chromosome walking
2. Probe again to identify clones containing the
end sequence of the first clone
sphere.bioc.liv.ac.uk8080/bio/studyweb/
modules/BIOL315/
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Chromosome walking

1. These clones must overlap the first clone.
   Hopefully they also contain some non-overlapping
   new DNA

sphere.bioc.liv.ac.uk8080/bio/studyweb/
modules/BIOL315/
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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.uk8080/bio/studyweb/
modules/BIOL315/
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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.uk8080/bio/studyweb/
modules/BIOL315/
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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.uk8080/bio/studyweb/
modules/BIOL315/
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• 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

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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

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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)
• One more critical ingredient

Then poison this recipe with small amounts of
dideoxy nucleotides to stop the reaction
Primer with 3 OH
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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
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Part of a DNA sequence
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BRCA1 found in 1994
Science. 1994 Oct 7266(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.
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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).
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Overview of PCR
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Overview of PCR
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Overview of PCR