chapter 7 analyzing dna and gene structure, variation and expression 1.sequencing and genotyping dna...
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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 non-coding 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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