利用AFLP技術探究缺氧對肝臟部份切除後再生之影響及基因表現之變化(3/3)
Date Issued
2003-07-31
Date
2003-07-31
Author(s)
李伯皇
DOI
912314B002164
Abstract
In this project, liver resection after a
period of ischemia in rats will be used as a
model to study the relation between ischemia
before hepatectomy and liver regeneration
after hepatectomy. And mRNA from the
samples will be extracted which stored in -70
℃ and double stranded cDNA will be
synthesized for amplified fragment length
polymorphism (AFLP) analysis.
To prepare an AFLP template, the
restriction fragments for amplifications are
generated by two different restriction
endonucleases. When used together, these
enzymes generate small cDNA fragments
that will be amplified well and are in the
optimal size range (<1Kb) for separation on
denaturing polyacrylamide gels. Following
heat inactivation of the restriction
endonucleases, the cDNA fragments are
ligated to adapters to generate template DNA
for amplification. PCR is performed in two
consecutive reactions.
In the first reaction, called
preamplification, cDNAs are amplified with
AFLP primers, each having one selective
nucleotide. The PCR products of the
preamplification reaction are diluted and
used as a template for the selective
amplification using two AFLP primers, each
containing three selective nucleotides. This
two-step amplification strategy results in
consistently cleaner and more reproducible fingerprints with the added benefit of
generating enough templates DNA for
thousands of AFLP reactions. Products from
the selective amplification are separated on a
denaturing polyacrylamide (sequencing) gel.
This technique would be taken
dissimilarity fragments among the samples
that could be reclaimed to sequence their
DNA sequences and similar comparing, so
we may understand how the genes expression
during these treatment.
Subjects
AFLP
liver
ischemia/reperfusion
Publisher
臺北市：國立臺灣大學醫學院外科
Type
report
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