Biomedical Engineering Reference
In-Depth Information
(a)
AGTTTTTTTT
GGTTTTTTTT
CGTTTTTTTT
TGTTTTTTTT
AATTTTTTTT
GATTTTTTTT
CATTTTTTTT
TATTTTTTTT
=
NVTTTTTTTT
ACTTTTTTTT
GCTTTTTTTT
CCTTTTTTTT
TCTTTTTTTT
(b)
AAAAAAAA
AAAAAAAA
AAAAAAAA
TTTTTTTT
AAAAAAAA
TTTTTTTT
N
V
N
V
N
V
N
V
TTTTTTTT
TTTTTTTT
N
9
N
9
N
V
T
TTTTTTT
AAAAAAAA
N
V
T
TTTTTTT
AAAAAAAA
(c)
Fig. 6.17
Summary of the differential mRNA display technique, after Liang and Pardee (1992). (a) A set of 12 oligo(dT) primers
is synthesized, each with a different two-base extension; the generic designation for this primer set is NVTTTTTTTTT, where N is
any nucleotide and V is any nucleotide except T. (b) Messenger RNA from two sources is then converted into cDNA using these
primers, generating 12 non-overlapping pools of first strand cDNA molecules for each source, The PCR is then carried out using
the appropriate oligo(dT) primer and a set of arbitrary 9-mers (N
9
), which may anneal anywhere within the cDNA sequence. This
facilitates the amplification of pools of cDNA fragments, essentially the same as expressed sequence tages (ESTs). (c) Pools of PCR
products, derived from alternative mRNA sources but amplified with the same pair of primers, are then compared side by side on a
sequencing gel. Bands present in one lane but absent from the other are likely to represent differentially-expressed genes. The
corresponding bands can be excised from the sequencing gel and the PCR products subcloned, allowing sequence annotation and
expression analysis, e.g. by northern blot or
in situ
hybridization, to confirm differential expression.
genes (Hubank & Schatz 1994). Essentially, the
technique involves the same principle as subtraction
hybridization, in that a large excess of a DNA from
one source, the driver, is used to make common
sequences in the other source, the tester, unclonable
(in this case unamplifiable). The general scheme is
shown in Fig. 6.18. cDNA is prepared from two
sources, digested with restriction enzymes and
amplified. The amplified products from one source
Enrichment for differences - representational
difference analysis
Representational difference analysis is a PCR sub-
traction technique, i.e. common sequences between
two sources are eliminated prior to amplification.
The method was developed for the comparative
analysis of genomes (Lisitsyn
et al
. 1993) but has
been modified for cloning differentially expressed
