Transcript Chapter 7 Analyzing DNA and gene structure, variation and

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.
07_01.jpg
07_02.jpg
07_02_2.jpg
• Automated DNA sequencing using fluorophores
and capillary gel electrophoresis.
07_03.jpg
• 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.
07_05.jpg
07_05_2.jpg
07_06.jpg
07_07.jpg
07_08.jpg
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.
07_09.jpg
• 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.
07_10.jpg
07_10_2.jpg
07_11.jpg
07_11_2.jpg
-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.
07_12.jpg
07_12_2.jpg
-iv- Mapping transcription start site could be
achieved by S1 nuclease protection or primer
extension.
07_13.jpg
3. Studying gene expression
• Principles of expression screening – in vitro
versus in vivo. RNA analysis versus tissues and
individual cells.
07_14.jpg
07_15.jpg
07_16.jpg
07_17.jpg
07_18.jpg
07_19.jpg