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 . . .)

12

4

8

2 , 8 4 , 8 1 , 2

1 , 8 2 , 4

Find markers that co-segregate with the disease

Restriction enzymes cut DNA at specific sites

Disease Allele “A”*

DNA probe

Normal Allele “B”DNA probe

AA AB BB

Different sequences will have different length fragments

BRCA1 is in the middle of Chromosome 17--What next?

Identifyrecombinants

Try moremarkers

Test more families

Recombination

246

453

121

864

243

864

Marker 1Marker 2Marker 3

Occasionally there is a crossover during meiosis

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?

Mapping BRCA1• 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

Chromosome 17

Figure 20.3

Restriction site

DNA 53 5

3G A A T T CC T T A A G

Sticky endFragment from differentDNA molecule cut by thesame restriction enzyme

One possible combination

Recombinant DNA molecule

G

C T T A AA A T T C

G

A A T T C

C T T A AG

G

G GA A T T C A A T T C

C T T A A G C T T A A G

Using a restriction enzyme and DNA ligase to make recombinant DNA

Cut DNA with Restriction enzyme, leaving overhanging ends

1

Base pairing of sticky ends produces various combinations.

2

DNA ligaseseals the strands.

3

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. sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

1b. Sequence the ends of that fragment.

2. Probe again to identify clones containing the end sequence of the first clone

Chromosome walking

sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

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

Chromosome walking

sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

4 Again, probe the large insert library to identify clones containing the sequence of the ends of these clones.

Chromosome walking

sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

4 Again, these clones must overlap the existing clones. ie they have some of the same DNA - and hopefully also some new sequence

Chromosome walking

sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

In this way we build up a CONTIG - a series of overlapping clones centred on our region of interest.

Chromosome walking

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)

+ One more critical ingredient:

Primer with 3’ OH

Then “poison” this recipe with small amounts of dideoxy nucleotides to stop the reaction

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

Di-deoxy sequencing

Part of a DNA sequence

BRCA1 found in 1994Science. 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.

Many different mutations in BRCA1 can lead to cancer

Results of sequencing

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