MCDB101Bslideset7

MCDB101Bslideset7 - 233 Molecular Tools for Genome...

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1 Molecular Tools for Genome Analysis (Chapter 9) Review material from 101A Restriction enzymes to cut DNA Gels to separate DNA/RNA by size Probes and hybridization to specifically detect complementary sequences Southern blots PCR to amplify miniscule amounts of DNA Libraries: genomic and cDNA DNA sequencing 233 Co In E Southern blot 234 Co In E
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2 Add denatured probe to nitrocellulose, let hybridize Nitrocellulose sheet in sealed bag probe molecules able to hybridize to DNA bound to the nitrocellulose 235 Co In E Human Bradykinin B1 Receptor Gene B – BamH1 E – EcoR1 H – HindIII K - KpnI exons probe Genomic Southern Blot 236 Genome Co In E
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3 Recombinant libraries A collection of clones that contain every sequence from the source nucleic acid Human genomic library: each clone in the library contains a single random fragment of human DNA cloned in a vector cDNA library: each clone contains a DNA copy of a single mRNA from the source tissue 237 Co In E Partial digests are used so enzymes cut only a subset of the total number of recognition sites in a genome Fig. 9.4 238 Co In E
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4 DNA Cloning Vectors 239 Co In E Fig. 9.7 Cloning genes in Plasmids 241 Co In E
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5 9.10 242 Co In E 9.11 243 Co In E
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6 Identifying Genes from Libraries • Detect epitope with antibody in expression library – requires antibody • purified protein • synthetic peptide • Colony or Plaque Hybridization – requires DNA probe • cDNA • Heterologous sequence • synthetic oligonucleotide • select populations based on differential expression 244 Co In E Fig. 9.12 Screening Expression Libraries 244a Co In E
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Hybridization is used to identify similar DNA sequences • Prepare library – Distribute libraries clones on petri dish – Transfer clones to nitrocellulose disk • Prepare probe – Previous cloned DNA – PCR fragment – Oligonucleotide • Screen library – Expose probe to clones on nitrocellulose – Determine location of matching clone by autoradiography or fluorescence Fig. 9.13 245 Co In E Characterization of Clones • Restriction Mapping • Genomic library – reconstruct map of chromosome by finding overlapping neighbor clones • DNA sequencing – Discover coding sequences, intron/exon junctions, regulatory elements, etc. 246
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MCDB101Bslideset7 - 233 Molecular Tools for Genome...

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