Download 2. Identifying coding sequences (genes)

Chapter 7
Analyzing DNA and gene structure,
variation and expression
1. Sequencing and genotyping DNA
•
Standard/manual DNA sequencing using
dideoxynucleotide chain terminators ddATP,
ddGTP, ddCTP, and ddTTP.
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• Automated DNA sequencing using fluorophores
and capillary gel electrophoresis.
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• Simple/basic genotyping by restriction site
polymorphisms (RSPs) and varaible number
tandem repeats (VNTRs)
- RSPs: single nucleotide polymorphism may
cause a loss or gain in a restriction site generating
an RSP. Used in identifying carriers for some
disease causing genes.
- VNTR: use of PCR or Southern blot
hybridization to identify differences in the number
of microsatellite tandem repeats.
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2. Identifying coding sequences (genes) in cloned
DNA (e.g. libraries) and establishing their
structure
• Three features distinguish coding DNA from noncoding DNA:
-i- coding sequences are highly conserved
-ii- presence, in coding sequences, of open reading
frames (ORFs).
-iii- vertebrate coding sequences are often
associated with CpG islands.
• Routine/traditional methods for identifying
evolutionary conserved coding sequences include
zooblots. Recently, homology searching of
sequence databases became a useful tool.
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• Besides routine methods, new more specialized
procedures are used to identify coding sequences:
-i- Exon trapping uses an artificial RNA splicing
assay.
-ii- cDNA selection by heteroduplex formation
using magnetic beads capture identifies expressed
sequences in genomic clones.
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-iii- To obtain full length cDNA of any gene, a
cDNA library is screened using a probe (an
oligonucleotide of the gene) and a set of
overlapping truncated cDNA clones are produced.
Then, RACE-PCR (rapid amplification of cDNA
ends) is a technique used to extend the 5’ and/or 3’
ends of a short cDNA clone to onbtain a full
length cDNA.
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-iv- Mapping transcription start site could be
achieved by S1 nuclease protection or primer
extension.
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3. Studying gene expression
• Principles of expression screening – in vitro
versus in vivo. RNA analysis versus tissues and
individual cells.
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