Chapter 7Analyzing DNA and gene structure, variation and expression • Sequencing and genotyping DNA • Standard/manual DNA sequencing using dideoxynucleotide chain terminators ddATP, ddGTP, ddCTP, and ddTTP.
Automated DNA sequencing using fluorophores and capillary gel electrophoresis.
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.
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.
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.
-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.
-iv- Mapping transcription start site could be achieved by S1 nuclease protection or primer extension.
3. Studying gene expression • Principles of expression screening – in vitro versus in vivo. RNA analysis versus tissues and individual cells.