Biomedical Engineering Reference
In-Depth Information
use the subtracted clone as a probe in Northern blots of mRNA from
cells A and B.
Differential display
Differential display is a PCR-based method to amplify differentially
expressed genes. mRNA from the two cell types to be compared is first
reverse transcribed into cDNA using oligo(dT) based primers, and then
these cDNAs are amplified with a set of short, random primers. The
bands generated by the primer sets from each cell type are then com-
pared on high resolution gels (denaturing polyacrylamide “sequencing”
gels are generally employed). The investigator then compares (literally
using the stare and compare method) the band patterns. Unique bands
found in one sample but not the other sample using the same sets of
primers, or bands represented at significantly different abundances, are
considered to be candidates for differentially expressed genes. To iden-
tify the mRNA represented by the candidate band, the band can be
sequenced and databases searched for the corresponding sequence
(the preferred method), or it can be excised from the gel and used to
probe cDNA libraries.
Representation difference analysis (RDA)
RDA is a method that combines subtractive hybridization with PCR
amplification to enrich for differences in genomes between two closely
related species or to enrich for differences in expressed genes between
two different tissues or cell types. Genomic DNA or cDNA from the two
DNA samples to be compared are digested with a restriction enzyme
that is a “frequent cutter”, and linkers are added to the DNA. Linkers are
short, double-stranded oligonucleotides that contain a restriction site.
In theory, PCR amplification using sequences in the linkers allows for
the unbiased amplification of the DNA samples, a critical step for sub-
tractive hybridization when comparisons are to be meaningful. Follow-
ing amplification, the linkers are then removed from both populations
of DNA. New linkers are added to one source of amplified DNA (the
tester). Small amounts of the tester are added to a large excess of the
second DNA (the driver) and melted (which generates denatured single
stranded DNA). The DNA is then allowed to hybridize. Only the DNAs
present in excess in the tester will rehybridize to itself, resulting in a small
subset of DNAs that have linkers on both strands since the excess driver
will hybridize with most of the tester DNA. This small subset can then
be amplified and cloned for analysis.
Search WWH ::
Custom Search