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CHAPTER-III
IDENTIFICATION AND ISOLATION
OF eDNA CLONES ENCODING TYROSINE-KINASES
FROM RAT SPLEEN eDNA LIBRARY
IDENTIFICATION AND ISOLATION OP eDNA CLONES ENCODING TYROSINE
KINASES PROM RAT SPLEEN eDNA LIBRARY
3.1. INTRODUCTION:
Amongst thousands of clones in a eDNA library, recombinant
clones of interest encoding a particular mRNA sequence are
usually identified and isolated by screening, which involves
rapid assays to determine whether a particular clone contains the
desired nucleic acid sequence.
The principle of screening recombinant libraries is based on
the fact that bacteriophage plaques or bacterial colonies
containing plasmids or cosmids contain relatively large amounts
of insert DNA which can be detected either directly by hybridiza
tion or indirectly by the protein that may be expressed from the
cloned segment.
The size of the library and the number of clones to be
screened reflects on the relative abundance of the mRNA of
interest. Since the abundance of the desired sequence is not
known with precision, it is usually preferred to select a library
having 5 times more recombinants than the total indicated by the
lowest abundance estimate. Generally the technique involves the
spreading of the library on agar or agarose plates. The clones
67
are then transferred to appropriate membrane filters and screened
using any one of the following methods:
1. Nucleic acid screening or hybridization to labelled DNA:
In this screening procedure recombinant DNA libraries are
screened by hybridizing the plaques or colonies which have been
transferred on to the filters to the nucleic acid probes which
have been radiolabelled or fluorescence labelled. Following a
prehybridization wash, the filter replicas are incubated with
denatured or single stranded DNA probe in appropriate hybridiza
tion buffer at appropriate temperature. The filters are then
washed to remove the excess probe and the positive clones are
identified either by autoradiography or fluorescence. (Grunstein
and Hogness 1975, Benton and Davis 1977).
2. The hybrid selection of mRNA and translation:
This method involves identification of cloned eDNA
complementary to specific mRNA by hybridization. The identified
clone is then denatured and immobilized on filters and hybridized
to mRNA preparation. The RNA is released from the RNA-DNA hybrid
by heating, and is translated in a cell-free in-vitro translation
system. The products are then identified by immunoprecipitation
and SDS-polyacrylamide gel electrophoresis, or by other
biological assays (Harpold et al., 1978).
3 • Expression screening or immunoreactivity:
Screening eDNA clones by hybrid-selected translation is
68
generally difficult when a rare mRNA is to be cloned or when the
efficiency of in vitro translation is low (eg. for large mRNA
molecules). These difficulties are overcome by cloning the eDNA
in bacterial expression vectors, and screening the antigenic
determinants expressed in situ using antibody which recognizes
the desired protein. The two basic technical procedures involved
in the immunological screening of bacterially synthesized fusion
proteins are, first, the synthesis and immobilization of the
antigenic materials to a solid support and second, detection of
antigen by a sensitive detection procedure. Both fusion products
and in vitro translated mRNA can be detected with the antibody.
In the present studies, eDNA clones encoding a tyrosine
kinase were identified and isolated from rat spleen eDNA
libraries in plasmid pGEM-3Z and bacteriophage Agtll using the
nucleic acid screening technique.
3.2. MATERIALS AND METHODS
3.2.1. MATERIALS:
Nitrocellulose filter papers were purchased from Schleicher . and Schuell, West Germany. The random primer labelling kit was
from Boehringer Mannheim, West Germany. The T4 polynucleotide
kinase was bought from Pharmacia, Sweden. PVP and salmon sperm
DNA were obtained from Sigma Chemical co., USA.
69
Description of the probes used:
A murine eDNA clone designated as bmk, a gift from Dr Ashley
R Dunn, Australia, was used as probe to identify the eDNA clones
coding for tyrosine kinases from the spleen eDNA library. It is a
1896 bp eDNA which has been subcloned into the Eco RI site of
pGEM 3 and the plasmid is called pM 41.5. The bmk clone shares
extensive homology with members of the src-related family of
protein tyrosine kinases. The myeloid and lymphoid cells of the B
cell lineage show high level of expression of this gene, and
hence has been named as bmk (B cell/myeloid kinase). It is also
expressed in normal macrophages. The bmk was found to have 86%
homology and similar expression pattern as compared with human
HCK, indicating that the bmk was the murine homologue of human
b£k. Therefore the term hck is used for both the loci (Holtzman
et al. , 1987) •
A 32-mer deoxyoligonucleotide for a region of the conserved
domain of the tyrosine kinases was synthesized. This probe was
used in order to identify eDNA clones of different tyrosine
kinases from the rat spleen eDNA libraries. The sequence of the
probe is shown below:
TTCAAGGGGTAGTTCACCTGTCGGGGACTCCG T A T
70
3.2.2. METHODS
Preparation of probes:
The plasmid pM 41.5 is a eDNA clone containing the 1896 bp
fragment of the bmk eDNA. The competent E.coli DH5a cells were
transformed with this plasmid as described in section
2.2.2. From a single colony of transformed bacteria a large scale
plasmid preparation was done using the procedure described by
Maniatis et al., (1982).
One litre culture of pM 41.5 plasmid was grown overnight in
LB containing 100p.g/ml ampicillin. The cells were collected by
centrifugation in a Sorvall RC5B centrifuge using GSA rotor at
6000rpm for 10min at 4°C, and washed with 200ml of cold STE (0.1M
NaCl, 10mM Tris-Cl pH 7. 8, 1mM EDTA) . The pellet was then
resuspended in 15ml solution I (50mM Glucose, 25mM Tris-Cl, pH
8.0, lOmM EDTA). To the suspended cells 5ml of solution I
containing 100mg lysozyme was added and mixed well with a glass
rod. Following incubation at room t~mperature for ~min, 40ml of
freshly prepared s.olution II ( 0. 2N NaOH, 1% SDS) was added and
mixed gently by inverting the tube several times and incubated
for lOmin on ice. Thirty ml of 5M potassium acetate, pH 4.8 was
then added, and the contents were mixed by quickly inverting the
tube several times and incubated for 10min on ice. The material
was centrifuged for 40min at 18000rpm in Sorvall RC5B centrifuge
at room temperature. The supernatant was decanted and the plasmid
was precipitated by adding o. 6 volumes of isopropanol and
incubating at room temperature for 15min. The plasmid DNA was
pelleted by centrifuging at 12000rpm for 30min at room
temperature and the pellet was washed with 70% ethanol, dried
under vacuum, and dissolved in 6ml TE. Further purification of
the plasmid DNA was carried out on cesium chloride - ethidium
bromide gradients. Solid cesium chloride was added to the DNA
solution in the ratio of 1gm per ml. To 10ml of the cesium
chloride plasmid solution 0.8ml of 10mg/ml ethidium bromide solu
tion was added. This solution was then poured into quickseal
centrifuge tubes and centrifuged in a Beckman L8-80 ultra
centrifuge, VTi-80 rotor, at 70,000 rpm for 5h 30min at 20°C. The
lower band of closed circular plasmid DNA was removed into a
syringe by inserting a 21 guage needle on the side of the tube.
The DNA solution was transferred to a siliconized glass tube and
the ethidium bromide was removed by repeated extractions with
equal volumes of water saturated 1-butanol until the pink colour
had disappeared. The aqueous phase was dialysed overnight against
several changes of TE (pH 8.0) •. The plasmid DNA .was precipitated
at -20°C after adding 0.1 volume of 3M sodium acetate (pH 5.2)
and 2. 2 volumes of ethanol. After keeping for 2h at -2o0 c the
plasmid was collected by centrifugation for 30min in an eppendorf
centrifuge. The pellet was dried briefly under vacuum and
dissolved in 2ml TE. The contaminating RNA was removed by treat
ing the DNA solution for 1h at room temperature with DNase free
RNase added to a final concentration of 10~gjml. The plasmid was
72
extracted once with phenol-chloroform, twice with chloroform and
precipitated with 0.1 volume of 3M sodium acetate and 2.2 volumes
ethanol at -2o0 c overnight. The precipitate was collected by
centrifugation at 12,000 rpm at 4°C. The pellet was washed with
70% ethanol, dried under vacuum and dissolved in 1ml TE. An
aliquot was analysed by electrophoresis on an 1% agarose gel to
check the preparation.
Electroelution of hck fragment:
About 50~g of pM 41.5 plasmid was digested with 100U Eco RI
and the 1896 bp hck fragment· was separated from the pGEM 3
plasmid fragment by electrophoresis in an 1% preparative agarose . gel. The hck fragment was purified by electroelution according to
the procedure of Maniatis et al., ( 1982) • Elution was done in
0.05X TBE in dialysis bag for 3h at 100V. The DNA solution was
collected and the fragment was precipitated by 0.1 volume of 3M
sodium acetate and 2.5 volumes of ethanol at -2o0 c overnight. The
fragment was electroeluted twice more to remove all contami-
nation with the vector pGEM-3.
Nick translation:
The probe was prepared by radiolabelling the hck fragment by
nick translation. The procedure for nick translation was as
described by Maniatis et al., ( 1982) • The reaction was carried
out in 50pl volume containing SOOng of DNA, 1 nmole of each un
labelled dNTPs, 20 pmoles of 32 P labelled dNTP (dATP or dCTP),
73
50mM Tris-Cl, pH 7.2, lOmM MgC1 2 , lOmM OTT, 2.5~g DNase free BSA,
50pg DNase I and 5 u of E.coli DNA Pol I. After 90min of incuba
tion at 15°C the reaction was stopped by adding 2pl of 0.5M EDTA.
The labelled DNA was purified from unincorporated dNTPs by spun
column chromatography.
Spun column chromatography:
Autoclaved Sephadex G-50 was poured in a 2ml disposable
plastic syringe till the top and packed by centrifuging for 5min
at JOOOrpm in HB 4 rotor in a Sorvall RC5B centrifuge. The nick
translated material was applied on top of the column. The tube
was washed with 100~1 of STE and the washings were also loaded on
the column. The eluate was collected in an eppendorf tube placed
beneath the syringe, by centrifugation at 4000rpm for 15min. The
percentage of incorporation of radioactivity was determined by
Cerenkov counting.
Random primed DNA labelling:
--.For some experiments the DNA fragment was labelled by the
random primed DNA labelling technique, using the kit obtained
from Boehringer Mannheim. The procedure followed was as
recommended by the supplier. The DNA was denatured by heating for
lOmin at 95°C and subsequently cooling on ice. The reaction
mixture in a final volume of 20pl contained lOOng of the
denatured DNA, 0.5nmol of each of unlabelled dNTPs, 20 pmoles of
labelled dNTPs, 2pl of solution containing hexanucleotide primer
74
in reaction buffer and 2 units of Klenow enzyme. Incubation was
at 37°C for 30min, after which the reaction was stopped by adding
lul of 0. 5M EDTA. The labelled fragments were separated from
unincorporated dNTPs by spun column seperation as described
above.
End labelling of deoxyoligonucleotide:
The 32 mer synthetic deoxyoligonucleotide was used for
screening after end labelling it with ( Y - 32 P) ATP using the
procedure of Maniatis et al., ( 1982) . The reaction was done in
20pl volume which contained, 250ng deoxyoligonucleotide, 2ul 10 x
Kinase buffer, 16.6 pmoles ( y-32 P) ATP, lOU of T4 polynucleotide
kinase. (10 x kinase buffer contains 0.5M Tris (pH 7.6), O.lM
Mgcl 2 50mM OTT, lmM spermidine, lmM EDTA). The mixture was incu
bated for 30min at 37°C and the reaction was stopped by adding
lpl of 0.5M EDTA. The probe was purified by spun column chromato
graphy as described earlier in this chapter. Sephadex G25 was
used instead of Sephadex G50.
Colony Hybridization:
Plating of colonies:
The plasmid library was plated on 48 soc agar plates (90mm
diameter) containing lOO}lg/ml ampicillin. On each plate about
5000 colonies were plated for high density screening. The plates
were incubated at 37°C for 17h. The transfer of colonies and
75
hybridization was done by the procedure of Grunstein and Hogness
(1975) with some modifications.
Transfer and lysis of colonies:
The plates with bacterial colonies were kept at 4°C for 2h.
For transfer 4 plates were removed each time. Nitrocellulose
filter (NC), 87mm diameter, was used for transfer straight from
the box. The dry NC was placed on the plate and orientation was
marked by stabbing a needle dipped in a nondiffusable ink through
the NC into the agar at asymmetric locations. The filter was
peeled off from the plate using a blunt ended forceps and placed
colony side up, for Jmin on two sheets of Whatman JMM filter
paper soaked in 10% SDS. The NC was transferred to a second set
of Whatman papers wetted with denaturing solution (0.5M NaOH,
1.5M NaCl) and left for 5min for colonies to lyse. Neutralizing
was done by placing them on a third set of Whatman)saturated with
neutralizing solution (1.5M Nacl; 0.5M Tris-Cl, pH 7.4) for 5min
and then for 5min on two Whatman sheets soaked in 2X SSPE. These
filters were then airdried for 30-60min and were placed between
Whatman JMM paper and baked for 2h at ao0 c in a vacuum oven. The
plates were reincubated at 37°C till the colonies appeared again.
They were then sealed and stored at 4°C.
Hybridization . . The filters were washed with 250m! of prewashing solution
(50mM Tris-Cl, pH 8.0·, 1M NaCl, lmM EDTA, 0.1% SDS) for 2h at
76
42°C. Prehybridization was then done for 6h at 65°C in 200m! of
prehybridization solution (5X SSPE, 5X Denhardts' solution, 0.1%
SDS, 100~g/ml denatured salmon sperm DNA).
Hybridization was done at 65°C for l8h. The double stranded
probe was denatured with 0.1 volume of 3N NaOH at 37°C for 5min
and added to 80ml prehybridization solution at a concentration of
5ngjml, and mixed well. The solution was transferred to two
crystallization dishes and the filters were immersed in it one by
one. In each dish 24 filters were placed and was shaken at 65°C
in a water bath.
The filters were washed at 65°C with 5X SSPE, 0.1% SDS for
30min, followed by 2 washes with 2X SSPE, 0.1% SDS for 30min each
and finally with 1X SSPE, 0.1% SDS for 30min and then for 15min.
The wet filters were arranged between plastic sheets and were
exposed to X-ray film at -70°C for 32h. The films were developed
and the positive colonies were replated at a density of about
100-300 colonies per 90mm plate for secondary screening. The
transfer and hybridization conditions were same as those used for
primary screening. After secondary screening positive clones were
picked up for further analysis.
The filter used for the primary screening were then rehybri
dized to the end labelled oligoprobe. Prehybridization and
hybridization were done at 37°c. The hybridization solution con
tained 3. 5ng of probe per ml. After hybridization for 20h the
77
filters were washed twice with 5 x SSPE, 0.1% SDS for 30min at
37°C and autoradiographed. The positive colonies were rescreened
using the same conditions.
Plaque Hybridization
Hybridization of plaques was similar to colony
hybridization. The Agt11 library of 80,000 plaques were screened
following the procedure of Benton and Davis (1977). E.coli Y1090
cells were infected with the phage as described in section
2..2.2. 18 plates were overlayed with the
incubated at 42°C for 10h. Each plate
> ¥-;i
infection mixture and
had about 4.5 x 103
plaques. The plates were cooled at 4°C for 2h. Transfer was done
by placing the NC on the plate for 30s. Using a needle dipped in
ink the filters were marked for orientation. The filters were
placed on 2 Whatman 3MM sheets soaked in denaturing solution. The
filters were neutralized, air dried and baked as described for
colony hybridization. The prewashing, prehybridization and hybri
dization were done at 65°C as was described for colony hybridiza
tion. The filters were exposed to x-ray films 14h at -7o0 c. The
positive clones were replated and rescreened using the same con
ditions of primary screening. The positive Agt11 clones were
picked up and DNA was prepared for further analysis.
Southern Hybridization:
The ~ositive clones obtained after secondary screening were
identified and picked up. Miniplasmid preparation was done with
the plasmid clones as described in section 2.2.2. DNA was prepa-
red from the positive clones of the Agtll library following the
procedure described in section 2.2.2, using the lysate obtained
from one 90mm plate. The plasmid DNA samples were digested with
EcoRI and analysed by electrophoresis on an 1% agarose gel. The
clones were digested with EcoRI and also double digested with
EcoRI and Hindiii and electrophoresed on a 0.5% agarose gel to
determine the size of the inserts. Southern hybridization of the
clones was performed to confirm the positive clpnes (Southern,
1975). The digested samples were seperated by electrophoresis on
an 1. 2% agarose gel at 7V/cm, till the bromophenol blue dye
reached 2cm from the edge of the gel. The gel was then denatured
for 30min by gently shaking it in denaturing solution. Neutrali-
zation was done for lh with two changes of neutralization solu-
tion. The gel was then washed with lOX SSPE and capillary
transfer was set up. Six pieces of Whatman 3 MM paper 0. Scm
larger than the gel were soaked in lOX SSPE and placed on a
plastic. ·sh·eet-. The·--·gel was placed on the stack of What-man .
filters. A nitrocellulose filter was cut to the exact size of the
gel and was soaked for Smin in 6X SSPE. It was put on top of the
gel without entrapping any air bubbles. All the edges of the
gel and NC were covered with Saran Wrap to prevent transfer of
buffer directly from the Whatman paper. Two sheets of Whatman
filter were cut exactly to the size of the gel, soaked in lOX
SSPE and placed on top of the nitrocellulose filter. A stack of
79
30-40 dry handmade filter papers were kept on top of the Whatman
• ~M • • paper, above wh1.ch a glass plateA_ we1.ghed down w1.th about SOOg
weight. Transfer was allowed to proceed overnight. In order to
ascertain complete transfer the gel was stained and examined
under uv. The DNA on the nitrocellulose paper was also seen by
keeping the NC on the UV transilluminator. The NC was air dried
for 30min and then baked under vacuum at 80°C for 2h.
Prehybridization and hybridization were done as described for
colony hybridization. Hybridization was for 16h in solution
containing 10 ng hck probe/ml. The nitrocellulose filters were
washed as described earlier. The final wash was with O.SX SSPE at
65°c. Autoradiography was done 14h at. -70°C.
3.3. RESULTS
Purification and labelling of the probe DNA:
The 1.89 kb EcoRl fragment of hck was used to screen the
eDNA libraries.The fragment was purified thrice by electro-
elution and was free from any contamination of the vector DNA.
The fragment was electroeluted three times to purify it from
contamination of the vector DNA. A dot blot of the plasmid with
nick translated hck fragment showed no hybridization of the
vector with the fragment (Fig.J.l) and hence the fragment could
be used for the screening of the eDNA libraries.
Radioactive labelling of the fragment was done either by
nick translation or by random primed labelling. The specific
80
1 2
A • B • c
Fig.3.1. Dot hybridization ofpGEM-3Z with electroeluted hck probe. Lane 1, pGEM-3Z; A, 20ng; B, lOng, C, 5ng; lane 2, adult rat spleen mRNA; A, 2pg; B. lpg; C, 0.5pg.
81
...
•
•
Fig.3.2. Primary screening of the spleen eDNA library in pGEM-3Z with hck probe.
81 Q.
activity of the probe was between 5 to 8 x 107 cpm per microgram
of DNA.
The 32-mer oligonucleotide was end labelled with ( y 32 P]
ATP. The incorporation was estimated by Cerenkov counting and
specific activity of the oligoprobe was calculated to be 4x1o7
cpm per microgram of probe.
Screening of plasmid eDNA library with hck fraqment:
The rat spleen eDNA library in plasmid pGEMJZ (unamplified)
was screened at high colony density, each plate having
approximately 5000 clones. A total of about 240,000 colonies were
screened and five regions of hybridization were detected
(Fig.3.2). Bacterial colonies from these positive regions were
removed and secondary screening at low density was performed.
Four colonies out of the five were positive on secondary
screening (Fig.3.3). Two positive colonies from each plate 1a,
1b, 3a, Jb, 4a, 4b, 5a, and 5b were picked and plasmids were
prepared from them. Analysis of the undigested plasmids on an 1%
agarose gel showed presence of inserts (Fig.3.4). Clones 1a, 1b,
3a, and 3b were larger than 4a, 4b, 5a and 5b. EcoR1 digestion
was done for 1a and 3b. The clones 1a and 3b were further
analysed by Southern hybridization. They hybridized to the ,bg
fragment and so were used for sequence analysis (Fig.3.5).
82
•
, •
Fig.3.3. Secondary screening of the spleen eDNA library in pGEM-3Z with hck probe.
83
123456789
Fig.3.4. Plasmid preparation of the hck positive clones of the plasmid library. Lane 1, pGEM-3Z; lane 2, 1a; lane 3, 1b; lane 4, 3a; lane 5, 3b, lane 6, 4a; lane 7, 4b; lane 8, Sa; lane 9, 5b.
1 2
Fig.3.5. Inserts released from the positive clones after EcoRI digestion. Lane 1, 1 a; lane 2, 3b.
84
Screening of the plasmid library with the oligonucleotide probe:
The filters which were used for primary screening with hck
fragment were directly screened with the end labelled oligo
nucleotide probe, without deprobing the filters. The autoradio
gram had 10 regions of faint hybri dization {Fig.3.6), which were
rescreened. Only one of the 10 clones showed positive hybridiza
tion after the secondary screeni ng (Fig.3.7). Plasmids were
prepared from 2 positive clones 6a and 6b. The clone 6b was
digested with EcoRl and analysed by electrophoresis on an 1%
agarose gel. It released an approximately 600 bp insert. This
clone was also analysed by sequencing.
Screening of the Aqtll eDNA library with hck fragment:
In order to isolate full lengt h clones, an unamplified rat
spleen eDNA library in ~t11 vector consisting of about 83,000
plaques {Table-IV, Chapter-!!) was s c reened with the b&k fragment.
After the hybridizatio~, 31 posit i ve plaques were identified
(Fig. 3. 8). Secondary screening was done for 10 of the strongly
hybridizing~laques. Nine filters had positive signal (Fig.3.9).
DNA was prepared from the positive clones L103, L108, L109, L110,
L111, L112, L115, and L116. Digestions were done with EcoR1 and
also double digestion with EcoR1 + Hindi!! (Fig. 2. 9 and 3 .10).
All the clones had released inserts. The EcoRl + Hindi!! digested
samples were analysed by Southern hybridization (Fig.3.11). The
inserts of all the clones were showing hybridization with the hck
fragment. Though positive clones were obtained from the plasmid
85
Fig.3.6. Primary screening of the spleen eDNA library in pGEM-3Z with deoxyoligonucleotide probe. The postive clone is shown encircled.
Flg.S.7. Secondi.lly screening of the spleen eDNA library in pla~ ..,. iJ pGEM-3Z wi!1 deoxyoligonucleotide probe.
86
Fig.3.8. Primary screening ofthe A.gtll spleen eDNA library with hck probe.
87
•
0
( 1/~ .r I •
,. . . I ' .
•
. . '
;'
Fig,3.9. Secondary screening of A.gtll spleen eDNA !ibrary with hck probe.
88
1 2 3 4 5 6 7 8 9 10
-23.1 kb
4.4
2.3 2.0
- 0.5
Fig.3.10. Inserts released after EcoRI +Hindiii digestion of the positive A.gtll clones. Lane 1, L103, lane 2, L108; lane 3, L109; lane 4, LllO; lane 5, Llll; lane 6, L112; lane 7, L115; lane 8, L116; lane 9, A.gt 11; lane 10, markers.
89
1 2 3 4 56 7 8
Fig.3.11. Southern hybridization of the A.gtll clones digested with EcoRI + Hindlll with hck probe. Lane 1, L116; lane 2, L115; lane 3, L112; lane 4, Llll; lane 5, LllO; lane 6, L109; lane 7, L108; lane 8, L103.
90
library the inserts were small in size. From the Agt11 library
more clones were obtained with larger insert size. These were
further analysed by sequencing to determine the presence of the
complete gene (Chapter-IV).
3.4. DISCUSSION
The screening of rat spleen eDNA libraries by two different
probes has given positive clones which are likely to code for
tyrosine-specific protein kinases {see also Chapter-IV) . This
shows that if appropriate probes are used for screening, positive
clones can be isolated even for a relatively rare mRNA such as
hck from the eDNA libraries. This also shows that the eDNA
libraries constructed (see Chapter-II) are in fact very good. The
plasmid eDNA library contains smaller cDNA·clones although same
preparation of eDNA was used for constructing plasmid as well as
Agt11 eDNA libraries {see Chapter-II). The smaller number of
positive clones obtained by screening plasmid library could be
due to the fact that the full length of hck gene is about 2 kb.
--The size selection in plasmid .eDNA library .. might .. have occurred at
either the ligation step or the transformation step or both. The
Agt11 eDNA library appears to be suitable for isolating full
length eDNA clones {size greater than 1 kb) whereas plasmid eDNA
library may be suitable for smaller inserts.
The oligonucleotide probe was designed to pick up src rela
ted tyrosine-specific protein kinase genes. It was expected to
91
pick up more clones than the hck probe since in addition to hck, . other src related kinase genes should hybridize with the probe.
Failure to obtain many positive clones with oligonucleotide probe
could be due to one or more of the several possible reasons (a)
design of the probe may be inappropriate (b) synthesis of the
probe may be inefficient and all possible oligomers may not be
represented (c) screening conditions may not be ideal.
The strategy of screening a eDNA 1 ibrary is of utmost
importance in any eDNA cloning project. At present several
different approaches can be attempted. Other oligonucleotide
probes can be designed for this purpose using some other
conserved region of the kinase domain. Another.· approach would be
to use the conserved kinase domain from one of the clones such as
Lll5 or Jb and screen a >. gtll library under less stringent
conditions followed by washing under stringent conditions. Using
this approach primary screening gave several positive clones most
of which are not hck clones since these clones do not appear
-"positive---under stringent- conditions of washing • Secondary screen-
ing and further analysis of these clones is under progress.
92