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Tools of human molecular genetics

Tools of human molecular genetics. Material in this lecture is largely review; we will cover technologies rapidly with emphasis on practical application Every term in the text box (“Language of Recombinant DNA Technology”) on p. 34 of text should be familiar to you as well as the usage examples

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Tools of human molecular genetics

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  1. Tools of human molecular genetics • Material in this lecture is largely review; we will cover technologies rapidly with emphasis on practical application • Every term in the text box (“Language of Recombinant DNA Technology”) on p. 34 of text should be familiar to you as well as the usage examples • Test yourself by doing the problems on pp. 49-50 (answers in back of book!)

  2. Molecular cloning resources and technologies • Restriction enzymes - cleave at specific sites, palindromes, “sticky” or blunt ends after cleavage • Ligation and vectors - plasmids, phage, cosmids, BACs, YACs (Table 4.2, p. 37) • DNA libraries - genomic, cDNA • Nucleic acid probes - cloned DNA or synthetic oligos

  3. “Instant” cloning - PCR • Requires primers, thermostable DNA polymerase • Extreme sensitivity is asset and a problem (contamination) • Many clinical and diagnostic applications

  4. Nucleic acid hybridization applications • Library screening - to isolate new sequences/probes • Southern blots - to analyze DNA sequences and polymorphic variations (e.g., RFLPs) • Northern blots - to analyze gene expression (e.g., to detect effect of mutation) • ASOs (allele-specific oligonucleotides) - to detect mutations

  5. “In situ” hybridization - ISH and FISH • Hybridization to chromosomal DNA denatured on microscope slide • Probes tagged with radioactivity (ISH) or, more commonly, with fluorescent tags (FISH) (e.g., Fig. 9-5A) • Chromosome paint probes and SKY (e.g., Fig. 9-5B) • Sperm aneuploidy evaluation (e.g., Fig. 9-5D) (Fig. 9-5 is between pp. 140 and 141)

  6. New “-omic” technologies • Genome-wide sequence analysis - “shot-gun” and ordered approaches with computational assembly • Global, genome-wide expression analyses - microarrays • Global whole-cell proteome analyses - mass spectrometry

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