1. What if we want to know what allele(s) of beta-globin an individual has?

2. Fig. 17-22 Wild-type hemoglobin DNA mRNA Mutant hemoglobin DNA mRNA 3 3 3 3 3 3 5 5 5 5 5 5 CCTT T T G G A A A A AA A GG U Normal hemoglobinSickle-cell hemoglobin Glu Val

3. Fig. 17-22 Wild-type hemoglobin DNA mRNA Mutant hemoglobin DNA mRNA 3 3 3 3 3 3 5 5 5 5 5 5 CCTT T T G G A A A A AA A GG U Normal hemoglobinSickle-cell hemoglobin Glu Val DdeI cuts: CTNAG

4. Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment 376 bp 201 bp 175 bp Dde I

5. Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragments Gel electrophoresis I II III Radioactively labeled probe for insulin gene DNA + restriction enzyme III Heterozygote II mutant insulin allele I Normal insulin allele Film over blot Probe detection Hybridization with radioactive probe Fragment from mutant Insulin allele Fragments from normal insulin allele Probe base-pairs with fragments Nitrocellulose blot 1 4 5 3 2

6. Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragments Gel electrophoresis I II III Radioactively labeled probe for insulin gene DNA + restriction enzyme III Heterozygote II mutant insulin allele I Normal insulin allele Film over blot Probe detection Hybridization with radioactive probe Fragment from mutant Insulin allele Fragments from normal insulin allele Probe base-pairs with fragments Nitrocellulose blot 1 4 5 3 2

7. Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment 376 bp 201 bp 175 bp Dde I Another option: PCR of Beta-globin gene, followed by DdeI digest

8. How can we measure gene expression? vs. wild typedif1 1.Isolate RNA 2.Compare gene expression

9. Fig. 20-13 TECHNIQUE RESULTS Gel electrophoresis cDNAs  -globin gene PCR amplification Embryonic stages Primers 1 2 3 4 5 6 mRNAs cDNA synthesis 1 2 3 Reverse Transcriptase PCR (RT-PCR)

11. 50 µm Where in the organism is my gene transcribed? Promoter : reporter fusions

12. Fig. 20-14 50 µm Where in the organism is my mRNA present? In situ hybridization

13. Fig. 20-15 TECHNIQUE Isolate mRNA. Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing specific genes DNA microarray with 2,400 human genes DNA microarray 1 2 3 4

14. WT dif1 ∆ dif1 myb98 ∆ myb98 genes Example of array data

15. TECHNIQUE Gel electrophoresis cDNAs PCR amplification Primers mRNAs cDNA synthesis 1 2 3 Reverse Transcriptase PCR (RT-PCR) Large scale sequencing of cDNA fragments Sequence large numbers (millions) of cDNA fragments

16. No UV (3 samples) UV (3 samples) Large scale sequencing of cDNA fragments Fragments matching rad51