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Bioscience Methods
BM 2011, Vol.2, No.6
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rations (Wan et al., 2002). However, there is no report
to reach this level of the concentration ratio so far.
Furthermore, endonucleases are employed in the
procedures of SSH for adaptor ligation, usually, it
might be uncertain whether or not the used endonuc-
leases could recognize all kinds of cDNA sequences,
which indicated that target genes should be missed in
the first step of SSH operation.
SSH normally uses mRNAwith poly-adenine (poly(A))
tails as template, therefore RNA without poly(A)
tails will be excluded in experimental steps that might
lose target genes. It is obvious that SSH might not
work well as exact as we expected when transcriptome
profiling is taken into account in the research.
In order to break above mentioned limitations, we
developed a modified method, named transcriptome
suppression subtractive hybridization (TSH) in
this research. The new method starts with the total
RNA from cells rather than mRNA only and no
endonuclease is employed at all in the THS.
To validate use reliability of the TSH, TSH was
employed to identify cell-specific expressing ESTs
in both
E. coli
JM109 with and without a recombinant
plasmid. The results showed that the non-target RNA
finally eliminated through direct hybridization and
digestion of RNA/cDNA hybrids. Six reference
genes within the recombinant plasmid were detected
as expected in the tester library constructed from the
JM109 RNA with the recombinant plasmid.
1 Results and Analysis
1.1 Screening cDNA libraries
Two cDNA libraries were constructed based on TSH
protocol. One library was generated by using cDNA
from JM109 that contained pC-SG after having done
subtractive hybridization with RNA from JM109
without pC-SG. This library was named JM109
+
cDNA library containing approximately 300 colonies.
While, the other library, named JM109
-
cDNA
library containing approximately 200 colonies was
cons-tructed by using cDNA from JM109 without
pC-SG, after having done subtractive hybridization
with RNA from JM109 with pC-SG.
Sixty-eight
colonies were randomly selected from
the
JM109
+
cDNA library. Each selected colony was
identified by colony PCR by using TSH primers. 37
positive colonies were identified out of the 68 colonies.
The results showed in figure 1 exhibited that the
sizes of the majority of the inserts varied from 200 bp
to x bp. Eighteen inserts from 37 positive colonies
were selected for sequencing.
The sequences were aligned by using the BLAST
online software to identify non-redundant sequences.
Thirty seven identified sequences represented fifteen
different transcripts. As shown in Table 1, 12 ESTs
Table 1 BLAST hits of ESTs from JM109
+
and JM109
-
cDNA library to
E. coli
K12 MG1655
Gene
Accession number
Annotation
wcaK
G7096
Pedicted colanic acid biosynthesis pyruvyl transferase
ygjK
G7599
Glycoside hydrolase
uvrC
EG11063
Excinuclease ABC subunit C
clpB
EG10157
Potein disaggregation chaperone
puuC
EG10036
Gma-aminobutyraldehyde dehydrogenase
mepA
EG10580
Pnicillin-insensitive murein endopeptidase
yraP
G7644
Lpoprotein
STSSU
DQ297414
Potato ADP-glc PPase small subunit
NPT
Ⅱ
AF354046
Nomycin phosphotransferase
Ⅲ
SCR
AF354046
Pasmid DNA replicase
HPT
Ⅲ
AF354046
Hgromycin phospho-transferase
ESTs from JM109
+
35S promoter
AF354046
CaMV 35S promoter
dmsB
EG10233
Dmethyl sulfoxide reductase, chain B
hslU
EG11881
ATPase component of HslUV protease
ESTs from JM109
-
ISIT
CU928160
ISI transposase