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

Fig Wild-type hemoglobin DNA mRNA Mutant hemoglobin DNA mRNA CCTT T T G G A A A A AA A GG U Normal hemoglobinSickle-cell hemoglobin Glu Val

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

Fig 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

Fig 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

Fig 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

Fig 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

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

Fig TECHNIQUE RESULTS Gel electrophoresis cDNAs  -globin gene PCR amplification Embryonic stages Primers mRNAs cDNA synthesis Reverse Transcriptase PCR (RT-PCR)

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

Fig µm Where in the organism is my mRNA present? In situ hybridization

Fig 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

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

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

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