Know thy selfLuxury Genes - from Forbes.com

Gift-hunting for a truly self-obsessed friend? Consider buying him/her a reading of his/her entire DNA code

A firm in Cambridge, Mass. called Knome (pronounced "gnome") is offering the

service.

http://www.knome.com/Recent News

October 2008: Knome Commences Whole-Genome Sequencing Process For First Clients

You, on a platter: A customer who pays $350,000 for Knome’s service receives a silver box containing an eight-gigabyte USB drive housing his or her genome sequence and specialized browser software. Credit: courtesy of Knome, Inc.

PCR Amplifies Target DNA

in vitro• Replicating target DNA in vitro • Taq polymerase

• Temperature-resistant DNA polymerase

– Thermus aquaticus•Heat resistant•Best for <2 kb target

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Fig. 9.16.4

PCR

Replicating target DNA in vitro2581b Hill C9

Fig. 9.16.1

•Two primers bracket the target region to be amplified•Complementary to one strand•Anti-parallel

Amplify DNA PCR

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Fig. 9.16.1

Amplify DNA PCR

The 3’ nucleotide is critical for specificity

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Fig. 9.16.3

Amplify DNA PCR

Cycles of repeated replication

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Fig. 9.16.2

Denature

AnnealExtend

Extend

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PrimerArtefact

Lane M 1 2 3 4 N P 5 6 7 8 9 10

1.8 kb

What is the size of the PCR product?

Gel electrophoresis separates PCR amplicons on the basis of size.

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• Exponential increase

• Need to know some sequence information

• Very sensitive

• Can amplify from limited starting material

• Ideal for 2kb target

PCR

Fig 8-8 2581b Hill C9

The PCR songhttp://www.youtube.com/watch?v=x5yPkxCLads&feature=related

Fig. 9.17.1

DNA sequencing

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Fig. 9.17.2

DNA sequencing

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Fig. 9.17.2

DNA sequencing

ddTTP

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Original Strand /Template strand

Nascent orNew Strand

Autoradiograph2581b Hill C9

Fig. 9.17.3

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Fig. 9.18.a

KH Genomics 10

Fig. 10.20.d

Automated DNA Sequencing

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Gels are now replaced by capillaries filled with polymers

Sequencing Chromatogram

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5’

3’Nascent or Newly synthesized strand

Close to the sequencing primer

Far from the sequencing primer

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Heterozygote Detection

Maternal templatesequence A

Paternal templatesequence C

SNPSingle nucleotide polymorphism

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Heterozygote Detection

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What would a chromatogram look like if the maternal copy had a deletion of one nucleotide?

A typical DNA sequencing read is 1000 nucleotides