Biology Reference
In-Depth Information
6.
Add 250
L of 70% ethanol, vortex briefly, and respin. Remove all traces of
ethanol with a pipet, if necessary respin sample to remove residual ethanol. Allow
pellet to air-dry for 10 min.
µ
7.
Electrophoresis of samples on ABI Prism 377 DNA sequencer (
see
Note 6
).
4. Notes
1.
In many cases, transformed lymphoblastoid (LB) cells are cultured in suspen-
sion. If this is the case, remove the medium and pellet cells by brief centrifuga-
tion. Resuspend cell pellet directly in TRIZOL.
2.
It is possible to use a gene-specific primer (GSP) in the first-strand cDNA syn-
thesis, we have found, however, that this is more likely to result in spurious prim-
ing in the PCR. We usually set up two separate RT reactions, which contain
oligo-dt and pd(N)
6
, respectively, and then pool these to give 40
L of cDNA for
PCR. In some cases, a DNA product is not seen after the first round of PCR.
There are several approaches that can be used to overcome this. The first is to
take an aliquot of the PCR reaction and reamplify using the same or preferably
nested PCR primers. Alternatively, it is possible to use more RNA in the RT
reaction or more cDNA in the PCR. In the latter case, it must be remembered that
there will be carry over of MgCl
2
and dNTPs, which will affect the specificity
of the PCR. Reduce proportionally the amount of MgCl
2
and dNTPs added to
the PCR to account for the carry over. A 10X PCR buffer can easily be made
without MgCl
2
, which is then added to the correct concentration from a 25-m
M
stock solution.
µ
3.
For DNA size determination, PAGE analysis of PCR products is far more accu-
rate than agarose gel electrophoresis. It is also possible to detect heteroduplex
DNA molecules on polyacrylamide gels (
Fig. 1
), which migrate slower com-
pared to homoduplex DNA molecules. Heteroduplex molecules are usually
formed when DNA is PCR amplified from a patient who is heterozygous for a
mutation. In the final stages of the PCR, double-stranded DNA molecules are
formed between normal and mutant single strands. This method of detection is
particularly suited for small deletions, which result in very distinctive heterodu-
plex bands. It is possible to accentuate the formation of heteroduplexes by one of
two ways. After the PCR has finished, heat the reaction to 96
C for 2 min then
allow to cool slowly to room temperature. Alternatively, the PCR reaction vol-
ume can be reduced to 25
°
L while using the same amount of DNA (100 ng), this
results in a more concentrated PCR product which will favor heteroduplex for-
mation, however, it may also result in spurious priming and nonspecific PCR
products.
µ
4.
Run the gel so that the nondenatured double-stranded DNA is about 2-3 cm from
the bottom of the gel (on a 6% PAGE, the xylene cyanol dye migrates at approx
300 bp). In this way, double-stranded heteroduplex molecules will be visible in
addition to the individual single strands (
Fig. 3
). By running the gel in the cold
room the voltage can be increased without the gel overheating and possibly dis-
rupting the secondary structure of the individual single stranded DNA molecules.
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