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Lecture 36: Cloningand SequencingGenes

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 GeneBRCA1

• 1980’s: found several families that werepredisposed 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 onChromosome 17q21– D17S74 - 183rd marker used!– Initial candidate region spanned half the

chromosome (hundreds of possible genes . . .)

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2 , 8 4 , 8 1 , 2

1 , 8 2 , 4

Find markers that co-segregate withthe disease

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Restriction enzymes cut DNAat specific sites

Disease Allele “A”*

DNA probe

Normal Allele “B”DNA probe

AA AB BBDifferent sequenceswill have differentlength fragments

BRCA1 is in the middle ofChromosome 17--What next?

Identifyrecombinants

Try moremarkers

Test morefamilies

Recombination

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453

121

864

243

864

Marker 1Marker 2Marker 3

Occasionally there isa crossover duringmeiosis

To find those rarecrossovers, theyneeded manyfamilies with inheritedbreast cancer

This individualshows that thedisease must notbe 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 theactual gene– Make a “library” of cloned fragments– Order those fragments– Find fragments that contain coding sequences– Sequence those fragments

Chromosome 17

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Figure 20.3

Restriction site

DNA 5′3′ 5′

3′G 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

GC T T A A

A A T T CG

A A T T C

C T T A AG G

G GA A T T C A A T T CC T T A A G C T T A A G

Using a restriction enzyme and DNAligase to make recombinant DNA

Cut DNA withRestrictionenzyme, leavingoverhanging ends

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Base pairing of sticky ends produces various combinations.

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DNA ligaseseals the strands.

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Note: The BRCA1study used YACsinstead ofplasmids, but theprinciples aresimilar.

Transformtherecombinantplasmid intoE. coli

To produce a “library” of different DNA fragments

One of the clones in the libraryshould contain the gene, but

which one?

1. Probe a large insertlibrary to identify aclone containing themarker linked to thetrait. sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

1b. Sequencethe ends ofthat fragment.

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2. Probe again toidentify clonescontaining the endsequence of the firstclone

Chromosome walking

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

3 These clones must overlap thefirst clone. Hopefully they alsocontain some non-overlappingnew DNA

Chromosome walking

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

4 Again, probe the large insert libraryto identify clones containing thesequence of the ends of these clones.

Chromosome walking

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

4 Again, these clones must overlap theexisting clones. ie they have some of thesame DNA - and hopefully also somenew sequence

Chromosome walking

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

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In this way we build up a CONTIG - aseries of overlapping clones centred onour region of interest.

Chromosome walking

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

• 8 cm may have many genes, but alsolots 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 containcoding sequences

• Make a DNA copy(“cDNA”) of themRNA usingReverseTranscriptase

• Use that to probefor clones thatcontain codingsequences

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Determining the NucleotideSequence

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” thisrecipe with smallamounts ofdideoxynucleotides to stopthe reaction

Precisely where thereaction stops eachtime is random, butif there are a millionnew strandssynthesized, eachpossible length offragment will beproduced

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 cancersusceptibility 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 MedicalCenter, Salt Lake City 84132.A strong candidate for the 17q-linked BRCA1 gene, which influencessusceptibility to breast and ovarian cancer, has been identified bypositional cloning methods. Probable predisposing mutations havebeen detected in five of eight kindreds presumed to segregate BRCA1susceptibility alleles.

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Many differentmutations inBRCA1 can leadto cancer

Results of sequencing

Now that the sequence is known, it is possible toamplify that region from other individuals, usingPCR (polymerase chain reaction).

Overview ofPCR

Overview ofPCR Overview of

PCR