Tools of human molecular genetics

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