BIOL 311 Human Genetics
Fall 2006
Recombinant DNA Methods: DNA Sequencing and Other Methods
Readings: See below
Outline:
1. dideoxy DNA sequencing
pp. 182-183
2. automated sequencing
3. other methods
a. S1 mapping pp. 189-190
b. primer extension pp. 189-190
c. bacterial expression vectors p. 147
d. Western blot p. 198, Fig. 7.17
e. eukaryotic expression vectors p. 150
f. gene transfer/eukaryotic cells pp. 151-154
g. pulsed field electrophoresis pp. 171-172
Lecture outline:
1. Dideoxysequencing
Key component:
Dideoxynucleotide (ddNTP): 2’, 3’ dideoxynucleotide is not able to extend growing
DNA chain; acts as chain terminator.
SeveraL HIV drugs ddI, ddC are dideoxy terminators—inhibit reverse transcriptase
activity of HIV virus.
DNA sequencing reaction requires:
 Single stranded DNA template
 Single stranded primer of 17-25 nts
 Mixture of deoxy and dideoxynucleotides, at least one is labeled
 Buffer
 A DNA polymerase such as T7 polymerase (manual sequencing) or Taq
polymerase (automated sequencing)
Automated DNA Sequencing: Genome Project Advances
1. multiplex reaction: reaction done in one tube, rather than four tubes
2. cycle sequencing: PCR with single primer forces reaction in one direction
3. fluorescent dyes attached to each of the dNTPs
1
4. slab gels replaced by capillaries
5. computer traces, not X-ray autoradiographs are the output
6. data can be immediately analyzed with bioinformatics software
7. competing commercial technologies (ABI, Beckman) with different dye chemistries,
different gel matrices
3. Other methods:
Techniques for identifying where transcription begins (transcription start site)
a. S1 mapping
Fig. 7-13a
Use γ-32P and polynucleotide kinase to create a labeled probe that includes location of
putative start site. Anneal probe to mRNA, then digest with S1 nuclease. Determine size
of protected fragment by gel electrophoresis.
Similar approach using riboprobe is called “RNase protection”
b. primer extension
Fig. 7-13b
End label DNA probe with kinase, digest ds DNA with restriction enzyme and purify
probe. Anneal probe to mRNA, add dNTPs and reverse transcriptase to synthesize DNA
strand. Determine size of synthesized product by gel electrophoresis.
c. bacterial expression vectors
 use to produce human protein in bacteria
 make large quantities for antibody production
Examples:
pET vectors: regulated expression of protein
GST vectors: regulated expression of fusion protein, affinity purification
d. Western blot
 use antibody to protein of interest to detect its presence in a mixture of proteins
separated by SDS-PAGE and transferred to a membrane.
i.e. detect dystrophin protein from muscular dystrophy patients, Fig. 7-17.
e. eukaryotic expression vectors



enable cloning in bacteria, expression of genes in eukaryotic cells
have features of bacterial cloning vectors, such as ampicillin resistance gene, pUC
origin of replication
have signals that enable expression of mRNA and protein in eukaryotic cells,
such as eukaryotic promoter (CMV promoter), poly A addition signal, introns,
dominant selectable marker (neomycin resistance)
2
f. gene transfer/eukaryotic cells
insert vectors or other DNAs into eukaryotic cells via any of several methods, especially
transfection
Methods: Ca phosphate precipitation, liposome transfer, electroporation, direct transfer
(microinjection)
g. pulsed field electrophoresis
modified agarose gel electrophoresis used to resolve very large DNA fragments from 20
kb to MB lengths
create fragments of DNA this large using rare cutter enzymes
Fig. 6.14 or animated version
Periodic switching of power between electrode pairs.
3