Transcript No Slide Title

Tools of human molecular
genetics
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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!)
Molecular cloning resources
and technologies
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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
“Instant” cloning - PCR
Requires primers, thermostable
DNA polymerase
 Extreme sensitivity is asset and a
problem (contamination)
 Many clinical and diagnostic
applications
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Nucleic acid hybridization
applications
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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
“In situ” hybridization - ISH
and FISH
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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)
New “-omic” technologies
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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