DNA Sequencing for Luxury Gift - KnowThySelfLuxury Genes

1. Know thy selfLuxury Genes - from Forbes.comGift-hunting for a truly self-obsessed friend? Consider buying him/her a reading of his/her entire DNA codeA firm in Cambridge, Mass. called Knome (pronounced "gnome") is offering the service. http://www.knome.com/Recent NewsOctober 2008: Knome Commences Whole-Genome Sequencing Process For First Clients

2. 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.

3. PCR Amplifies Target DNAin vitro • Replicating target DNA in vitro • Taq polymerase • Temperature-resistant DNA polymerase • Thermus aquaticus • Heat resistant • Best for <2 kb target 2581b Hill C9

4. Fig. 9.16.4 PCR Replicating target DNA in vitro 2581b Hill C9

5. Fig. 9.16.1 Amplify DNA PCR • Two primers bracket the target region to be amplified • Complementary to one strand • Anti-parallel 2581b Hill C9

6. Fig. 9.16.1 Amplify DNA PCR The 3’ nucleotide is critical for specificity 2581b Hill C9

7. Fig. 9.16.3 Amplify DNA PCR Cycles of repeated replication 2581b Hill C9

8. Fig. 9.16.2 Denature Extend Anneal Extend 2581b Hill C9

9. Lane M 1 2 3 4 N P 5 6 7 8 9 10 1.8 kb Primer Artefact What is the size of the PCR product? Gel electrophoresis separates PCR amplicons on the basis of size. 2581b Hill C9

10. PCR • The PCR song • http://www.youtube.com/watch?v=x5yPkxCLads&feature=related • Exponential increase • Need to know some sequence information • Very sensitive • Can amplify from limited starting material • Ideal for 2kb target Fig 8-8 2581b Hill C9

11. Fig. 9.17.1 DNA sequencing 2581b Hill C9

12. Fig. 9.17.2 DNA sequencing 2581b Hill C9

13. 2581b Hill C9

14. Fig. 9.17.2 DNA sequencing ddTTP 2581b Hill C9

15. Original Strand / Template strand Nascent or New Strand Autoradiograph 2581b Hill C9

16. Fig. 9.17.3 2581b Hill C9

17. Fig. 9.18.a KH Genomics 10

18. Fig. 10.20.d Automated DNA Sequencing Gels are now replaced by capillaries filled with polymers KH Genomics 10

19. Sequencing Chromatogram 5’ Close to the sequencing primer Far from the sequencing primer 3’ Nascent or Newly synthesized strand KH Genomics 10

20. Page 349 Heterozygote Detection Maternal template sequence A Paternal template sequence C SNP Single nucleotide polymorphism KH Genomics 10

21. Page 349 Heterozygote Detection What would a chromatogram look like if the maternal copy had a deletion of one nucleotide? A typical DNA sequencing read is 1000 nucleotides KH Genomics 10