The document discusses DNA sequencing techniques, specifically the Sanger method of DNA sequencing. It describes how the Sanger method uses dideoxynucleotides (ddNTPs) which can be incorporated into the growing DNA strand during replication but prevents further elongation. Short primers are used to hybridize and prime DNA synthesis. Microarray analysis is also summarized as measuring the gene expression levels of many genes by isolating mRNA, making cDNA, hybridizing the cDNA to an array chip containing short sequences of many genes.

1. 12a DNA sequencing: the Sanger method Nobel prize to David Sanger Use of dideoxy-nucleotides or ddNTPs ddNTP can be incorporated but not elongated

2. Primer: Short (15-20) string of dNTPs Hybridizes to complementary strand Primes DNA synthesis (Ch 16!) 12b DNA sequencing: the Sanger method 3’ 5’ 3’ 5’ Taq 5’ 3’

3. 12c DNA sequencing: the Sanger method

4. 12c DNA sequencing: the Sanger method

5. 12c DNA sequencing: the Sanger method

6. 12c DNA sequencing: the Sanger method Alberts 4 th ed 8.37

7. 12c DNA sequencing: the Sanger method Alberts 4 th ed 8.37

8. 15 Micro-array analysis Measures gene expression levels of many genes Procedure Isolate mRNA Cells, tissues, tumors,.. Make cDNA Using fluorescent nucleotides Hybridize cDNA with array-chip Chip contains short sequence of many genes Primers PCR-products …

9. 15 Micro-array analysis Measures gene expression levels of many genes Procedure Isolate mRNA Cells, tissues, tumors,.. Make cDNA Using fluorescent nucleotides Hybridize cDNA with array-chip Chip contains short sequence of many genes Primers PCR-products …

10. 15 Micro-array analysis Measures gene expression levels of many genes Procedure Isolate mRNA Cells, tissues, tumors,.. Make cDNA Using fluorescent nucleotides Hybridize cDNA with array-chip Chip contains short sequence of many genes Primers PCR-products …

11. 15 Micro-array analysis Measures gene expression levels of many genes Procedure Isolate mRNA Cells, tissues, tumors,.. Make cDNA Using fluorescent nucleotides Hybridize cDNA with array-chip Chip contains short sequence of many genes Primers PCR-products …

12. 15 Micro-array analysis Measures gene expression levels of many genes Procedure Isolate mRNA Cells, tissues, tumors,.. Make cDNA Using fluorescent nucleotides Hybridize cDNA with array-chip Chip contains short sequence of many genes Primers PCR-products …

13. Only briefly mentioned techniques But very important Not tested for these in exam! Knock-out In vitro mutagenesis RNA interference Transgenic animals Homologous recombination SNPs STR RFLPs

14. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair Procedure Isolate donor cell Isolate nucleus Isolate oocyte Remove DNA from egg Inject nucleus into egg Grow Implant Wait

15. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair Procedure Isolate donor cell Isolate nucleus Isolate oocyte Remove DNA from egg Inject nucleus into egg Grow Implant Wait

16. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair Procedure Isolate donor cell Isolate nucleus Isolate oocyte Remove DNA from egg Inject nucleus into egg Grow Implant Wait

17. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair Procedure Isolate donor cell Isolate nucleus Isolate oocyte Remove DNA from egg Inject nucleus into egg Grow Implant Wait

18. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair Procedure Isolate donor cell Isolate nucleus Isolate oocyte Remove DNA from egg Inject nucleus into egg Grow Implant Wait

19. 20 Stem cells Very hot issue Medical applications

20. 20 Stem cells Very hot issue Medical applications

21. 20 Stem cells Very hot issue Medical applications

22. 20 Stem cells Very hot issue Medical applications

23. 22 Gene therapy DNA technology to correct genetic disorders Especially suitable for blood disorders Proven correction in 2002! Procedure Clone gene Insert in virus Isolate cells Grow cells Infect cells Select right cells Inject corrected cells

24. 22 Gene therapy DNA technology to correct genetic disorders Especially suitable for blood disorders Proven correction in 2002! Procedure Clone gene Insert in virus Isolate cells Grow cells Infect cells Select right cells Inject corrected cells

25. Diagnosis of disease Made possible by DNA technology: PCR Detect virus RNA or DNA in serum: HIV! Detecting abnormal forms of genes SNPs Single nucleotide polymorphism STR analysis RFLP: Huntington DNA sequencing

26. 25 Transgenic organisms Genetic engineering of bacteria Cleaning water Sheep, goats Protein in milk Plants Relatively easy one cell will develop into plant

27. Golden rice Genes for synthesis of ß-carotene inserted into rice DNA Vitamin A deficiency

28. Summary of the techniques DNA analysis Electrophoresis Sequencing Southern blotting RFLP Practical applications Diagnosis of disease Gene therapy Fingerprints-RFLP Genetic engineering DNA cloning Restriction enzymes Recombinant DNA Cloning a gene into a plasmid DNA libraries PCR