U.S. patent application number 10/428541 was filed with the patent office on 2003-10-30 for method for the 5'-cap-dependent amplification of cdnas.
This patent application is currently assigned to Roche Diagnostics Corporation. Invention is credited to Mueller, Manfred W., Schmidt, Wolfgang M..
Application Number | 20030203389 10/428541 |
Document ID | / |
Family ID | 27614456 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203389 |
Kind Code |
A1 |
Mueller, Manfred W. ; et
al. |
October 30, 2003 |
Method for the 5'-cap-dependent amplification of cDNAs
Abstract
The invention concerns a method for the modification, cloning
and amplification of cDNAs which are complete at their 5' end which
is essentially characterized in that the first strand cDNA
synthesis is carried out in the presence of manganese.sup.2+ ions
or manganese.sup.2+ is added as an additive at a later time. The
CAP structure at the 5' end of the reversely transcribed mRNA
triggers the attachment of deoxy-cytosines to the 3' end of the
cDNA with high efficiency. In a preferred embodiment a controlled
ribonucleotide tailing is carried out with the aid of terminal
transferase following the first strand cDNA synthesis.
Inventors: |
Mueller, Manfred W.;
(Klosterneuburg, AT) ; Schmidt, Wolfgang M.;
(Wien, AT) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Assignee: |
Roche Diagnostics
Corporation
|
Family ID: |
27614456 |
Appl. No.: |
10/428541 |
Filed: |
May 1, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10428541 |
May 1, 2003 |
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09566794 |
May 5, 2000 |
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6558927 |
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Current U.S.
Class: |
435/6.16 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6855 20130101;
C12Q 1/6855 20130101; C12Q 2521/131 20130101; C12Q 2521/107
20130101; C12Q 2527/125 20130101; C12N 15/1096 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
May 5, 1999 |
DE |
19920611.2 |
Claims
What is claimed is:
1. A method for the modification, cloning or amplification of a
cDNA, wherein the first strand cDNA synthesis is carried out in the
presence of magnesium.sup.2+ ions and manganese.sup.2+ ions.
2. A method for the modification, cloning or amplification of a
cDNA, wherein the cDNA is incubated in the presence of
manganese.sup.2+ ions directly after the first strand cDNA
synthesis.
3. The method of claim 1, wherein the concentration of
manganese.sup.2+ ions is at least 1 mM but at most 20 mM during the
cDNA synthesis.
4. The method of claim 1, wherein after the cDNA synthesis, the
cDNA is reacted with a ribonucleotide triphosphate or a modified
nucleotide triphosphate in the absence of terminal transferase.
5. The method of claim 4, wherein the product of the reaction is
linked to a further double-stranded nucleic acid molecule which has
a 3' over-hanging end and is complementary to the 3' end of the
product of the reaction.
6. The method of claim 5, wherein the further nucleic acid molecule
is a DNA vector or an adaptor molecule.
7. The method of claim 5, wherein the further nucleic acid molecule
is ligated to the 3' end of the product of the reaction.
8. The method of claim 7, wherein the ligation is carried out in
the presence of 5 to 10 vol % DMSO.
9. The method of claim 8, wherein the specific ligation is carried
out with an efficiency of more than 90%.
10. The method of claim 7, wherein the amplification is carried out
using a primer provided with an immobilizable group.
11. The method of claim 7, wherein the double-stranded DNA adaptor
molecule is provided with an immobilizable group.
12. The method of claim 10, wherein the amplification reaction is
carried out several times.
13. The method of claim 7, wherein a nested PCR is carried out.
14. The method of claim 10, wherein the amplification product is
directly sequenced.
15. A mixture of double-stranded DNA adaptors containing 3'
overhangs composed of 5'-dT.sub.3-4dG.sub.3-3'.
16. First strand cDNA characterized by a non mRNA template-coded 3'
end composed of 5'-dC.sub.3-4rA.sub.3-4-3'.
Description
[0001] The present application claims priority to co-pending German
Patent Application No. 19920611.2, filed May 5, 1999, which is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention originates from the field of cloning and
amplification of nucleic acids and concerns a method for cloning
cDNAs that are complete at the 5' end.
BACKGROUND OF THE INVENTION
[0003] The molecular analysis of messenger RNAs (mRNAs) that have
been transcribed in vivo is usually carried out by generating and
cloning so-called cDNAs. For this a previously isolated,
poly-adenylated mRNA is firstly reversely transcribed using an
oligo-dT primer i.e. it is transcribed into a single-stranded cDNA
which is complementary to the mRNA. Subsequently a second strand
which is complementary to the single-stranded cDNA is polymerized
by methods known to a person skilled in the art to form a
double-stranded cDNA. After amplifying and cloning the cDNA or
parts of the cDNA using conventional molecular-biological methods
(cf. Sambrook, Molecular Cloning, Laboratory Manual, 2nd edition,
chapter 14), it is possible to subsequently determine the sequence
of the mRNA. However, a disadvantage of this method is that it is
seldom possible to identify and clone cDNAs which have a complete
5' end or can only be achieved with a very low efficiency. The
reason for this is an inefficient reverse transcription reaction
due to formation of intramolecular secondary structures as well as
a limited amount of starting material whose RNA content is of
inadequate quality. An additional disadvantage is that a loss of
terminal 5' sequences of the mRNA occurs in conventional methods
due to the manner in which the second strand is synthesized.
[0004] Various methods which have been developed in the past to
overcome this problem have concentrated on using the cap structure
of the mRNA which occurs at the 5' end of complete cDNAs as an
additional selection criterion. This is a terminal guanosine
residue which is methylated at position 7 and is linked by a 5'-5'
bond to the actual mRNA.
[0005] In the oligo capping method (Maruyama and Sugaru, Gene 138,
171-174, 1994) the cap structure is firstly removed enzymatically
by a suitable phosphatase treatment and replaced by an
oligonucleotide which is linked to the 5' end of the mRNA by a
suitable ligation step. However, this method requires many
enzymatic steps and a large amount of poly-A-mRNA as the starting
material.
[0006] Another method, the Cap Finder method (Clontech,
Clontechniques 11, 1 (1996), Maleszka and Stange, Gene 202, 39-43
(1997)) is based on a marginal terminal transferase activity of the
reverse transcriptase which leads to a so-called template switching
effect: In a first strand cDNA synthesis a few deoxy-cytosine
residues are added selectively to the 3' end of the cDNA by the
reverse transcriptase under suitable conditions. This produces an
anchor sequence for a so-called template switching oligonucleotide
with a 3' end composed of guanosine residues which, after
hybridization to the anchor sequence, serves as a primer for the
second strand synthesis of the cDNA. However, the terminal
transferase activity of the reverse transcriptase which apparently
begins at the 7-methyl-guanosine-cap structure of the mRNA and adds
the cytosine residues, has previously not been characterized in
detail so that the influence of certain secondary structures of the
mRNA template or of the cDNA end on the template switching activity
was unknown at the time of the invention and according to the prior
art the reaction could only be carried out with a low
efficiency.
[0007] In an alternative method (WO 97/26368) a suitable anchor
sequence is synthesized with the aid of a terminal transferase
enzyme which is different from the reverse transcriptase in the
process of which two to four ribonucleotides (instead of
deoxyribonucleotides) are attached as an anchor sequence to the 31'
end of the first strand cDNA. However, a disadvantage of this
method is that the cDNA synthesis cannot be carried out selectively
for 5'-cap mRNAs.
SUMMARY OF THE INVENTION
[0008] Hence the technical object of the invention was to develop
an additional method for the modification, cloning or amplification
of cDNAs which is used to obtain cDNAs that have a complete 5' end
in the simplest possible manner and as efficiently as possible.
[0009] This technical object is achieved in that the terminal
transferase reaction that elongates the first strand cDNA with
deoxy-cytosines is carried out by a reverse transcriptase in the
presence of magnesium.sup.2+ ions as well as in the presence of
manganese.sup.2+ ions. The manganese concentration to be used is
preferably 1-20 mM and under optimized conditions 8 mM.
[0010] In one embodiment the manganese.sup.2+ ions can already be
contained in the buffer system used during the cDNA first strand
synthesis. Alternatively an incubation in the presence of
manganese.sup.2+ ions can be carried out directly after a first
strand cDNA synthesis carried out in the absence of
manganese.sup.2+ ions in which case the reverse transcriptase used
originally is still active under these conditions.
[0011] In this process the reverse transcriptase develops a
terminal transferase activity which leads to an efficient addition
of 2 to 4 deoxy-cytosine residues at the 3' terminus of the newly
synthesized cDNA. This reaction is extremely efficient and also
dependent on the presence of the cap structure at the 5' end of the
mRNA template.
[0012] In both embodiments the deoxy-nucleotide tailing in the
presence of Mn.sup.2+ ions according to the invention not only
results in a high efficiency of the tailing reaction. At the same
time the specificity of the reaction is retained i.e. the addition
of two to four deoxy-cytosine residues in the presence of a 5'-cap
structure on the mRNA template.
[0013] Any reverse transcriptase enzymes can be used with the only
restriction that the enzyme that is used should not have any RNAseH
activity.
[0014] A so-called "anchor primer" is preferably used as the primer
for the first strand synthesis which is composed of two parts: a
so-called "anchor sequence" is located at the 5' end which can
serve as a target sequence for PCR primers in amplification
reactions that take place at a later time. In contrast the 3'
terminal end is composed of a sequence that can hybridize with the
mRNA to be identified. This can for example be an oligo-dT sequence
which hybridizes with the poly-A tail 3' end of the mRNA.
[0015] Following a cDNA synthesis according to the invention, the
first strand cDNA is reacted in a preferred embodiment with a
ribonucleotide triphosphate or comparable derivatives such as
2'-O-methyl or 2'-O-amino nucleotide triphosphates in the presence
of a terminal transferase that is different from reverse
transcriptase to form an anchor sequence of ribonucleotide residues
at its 3' end. When a certain ribonucleotide triphosphate is used,
the reaction is referred to as controlled ribonucleotide tailing
(CTRT). The ribonucleotide is preferably not CTP; the use of ATP is
particularly advantageous.
[0016] The present invention also concerns methods in which the
product of the reaction is subsequently linked to an additional
double-stranded nucleic acid molecule which has a 3'-overhanging
end that is complementary to the 3' end of the product of the
reaction. Suitable additional double-stranded nucleic acid
molecules are for example DNA vectors, or short adaptor molecules
which have target sequences for PCR primers for the subsequent
amplification.
[0017] Furthermore the double-stranded nucleic acid molecules that
are used can contain sequences which facilitate a subsequent
analysis after the amplification is completed. These for example
include promoter sequences suitable for in vitro transcription such
as the prokaryotic T7, T3 or SP6 promoters and also restriction
cleavage sites of which rare cleavage sites for so-called rare
cutter enzymes such as NotI are particularly preferred.
[0018] The additional double-stranded nucleic acid molecule is
preferably ligated to the 3' end of the product of the above
mentioned reaction. In this connection it has turned out that
hybridization of the adaptor to the first strand cDNA-is
particularly efficient in the presence of standard ligation buffers
containing DMSO and efficiencies of more than 90% can be achieved.
Concentrations of 5-10 vol. % DMSO have proven to be advantageous.
A concentration of 7.5 vol % was determined during the optimization
of the reaction.
[0019] This step includes the actual selection of full-length cDNA
molecules which have the specific 3' overhangs as a result of the
selection of adaptors or vectors. These overhangs are essentially
characterized in that they are completely complementary to the 3'
end of the initially synthesized full-length cDNA. This is
accomplished by the overhang being composed of 3 or 4
deoxy-thymidine residues followed by 3 deoxy-guanosine. residues
when ATP is used for the terminal transferase reaction.
[0020] Due to the fact that the number of nucleotide residues added
to each cDNA molecule during the tailing reaction of the reverse
transcriptase and during the controlled ribonucleotide tailing is
not completely identical, it has proven to be advantageous when the
adaptors are composed of a mixture of molecules whose 3' overhangs
vary between 3 and 4 deoxy-thymidine residues and possibly also
between 3 and 4 deoxy-guanosine residues.
[0021] Consequently a mixture of double-stranded DNA adaptors
containing 3' overhangs composed of 5'-dT.sub.3-4dG.sub.3-3' is
also a subject matter of the invention. The invention also concerns
single-stranded cDNAs which have a non-mRNA template coded 3' end
composed of 5'-dC.sub.3-4-rA.sub.3-4-3'.
[0022] In the subsequent amplification according to the invention
using PCR it is possible to distinguish between several basic
methods:
[0023] A) Construction of a cDNA Gene Bank
[0024] A primer pair comprising an adaptor primer and a so-called
anchor primer is used for this. As described above the adaptor
primer that is used is directed against the double-stranded
sequence of the DNA adaptor molecule. In contrast the sequence of
the anchor primer corresponds to the anchor part that was attached
to the 5' end of the cDNA during the first strand cDNA synthesis by
the reverse transcriptase reaction. The pool of amplification
products formed in this manner potentially contains numerous
different full-length cDNAs and can be used to generate a cDNA gene
bank by conventional molecular biological methods.
[0025] FIG. 1 shows a schematic overview of the method according to
the invention: The method combines the 5' CAP-dependent addition of
3-4 cytosine residues to the 3' terminus of full-length first
strand cDNAs by the reverse transcriptase (RT) (1., with the
inventive use of a reaction buffer containing manganese chloride)
with the technique of controlled ribonucleotide tailing of cDNA
ends (CRTC) by the enzyme terminal transferase and rATP (2.). The
terminal sequence motif (5'-dC.sub.3rA.sub.3-4) formed in this
manner enables the full-length cDNA to be selectively ligated to a
double-stranded DNA adaptor (5'-dT.sub.3-4dG.sub.3) using T4 DNA
ligase (3.). This method leads to the specific amplification of
full-length cDNAs which contain a complete 5' sequence of the mRNA.
Subsequently a cDNA bank can be constructed (5.2.) after a suitable
amplification by PCR.
[0026] B) Isolation and Amplification of a Specific mRNA for which
Partial Sequence Information is Available.
[0027] In this step a so-called adaptor primer and an mRNA primer
are used as the primers for amplification. The adaptor primer is
directed against the double-stranded region of the DNA adaptor
molecule. In contrast the mRNA primer is complementary to a
previously known sequence section of the mRNA. The amplification
products obtained in this manner represent cDNAs which are complete
at their 5' end but have a sequence section of a particular length
missing at their 3' end that depends on the choice of the mRNA
primer.
[0028] If the mRNA to be identified is a weakly expressed species,
it can be amplified again in a nested PCR using an additional
primer pair of which one primer is in turn directed against the
double-stranded adaptor sequence and an additional primer is
directed against an internal sequence of the mRNA.
[0029] In a special embodiment the adaptor primer can contain an
immobilizable label such as biotin so that the amplification
products that are formed can be bound to a solid phase coated with
streptavidin. In this manner the amplification products can for
example be more simply analysed by direct sequencing.
[0030] The invention additionally concerns methods in which the
cDNA is amplified using a primer provided with an immobilizable
group. In an alternative embodiment the double-stranded DNA adaptor
molecule is, by contrast, provided with an immobilizable group. As
a result the amplification product, whose generation is dependent
on cDNA, can be coupled to a solid phase for further analysis.
[0031] The invention additionally concerns in particular methods
which are characterized in that the amplification reaction is
carried out several times. This repeated amplification can be
carried out with several amplification primers in the form of a
nested PCR.
[0032] The invention also concerns particular embodiments of the
inventive method in which the amplification product is subsequently
directly sequenced.
[0033] In addition to the said methods, other amplification methods
are also a subject matter of the invention which are based on
special analytical formats such as differential display (WO
93/18176), serial analysis of gene expression (U.S. Pat. No.
5,695,937), subtractive hybridization (Wang et al., Proc. Natl.
Acad. Sci. USA 88:11505 (1991)) or linear amplification with the
aid of T7 RNA polymerase.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1: overview scheme of the method for cap-selective
CRTC
[0035] FIG. 2: first strand cDNA synthesis: 5'-CAP-dependent
terminal transferase activity of a reverse transcriptase in the
presence of manganese chloride
[0036] FIG. 3: 5'-CAP-dependent terminal transferase activity with
various reverse transcriptases
[0037] FIG. 4: manganese-dependency of the terminal transferase
activity of reverse transcriptase
[0038] FIG. 5: controlled ribonucleotide tailing with rATP
[0039] FIG. 6: selective ligation of full-length cDNA
[0040] FIG. 7: inventive amplification with the aid of PCR
[0041] FIG. 8: direct sequencing of inventive amplified cDNA
EXAMPLES
Example 1
[0042] 5'-CAP-dependent Terminal Transferase Activity of the
SuperScript .TM. RT
[0043] The terminal transferase activity of the
SuperScript.upsilon.RT (Gibco BRL) was tested under the following
conditions using an in vitro transcribed 226nt RNA (.beta.-globin
from the rabbit, GenBank V00882, fragment (pos. 633-1335) cloned in
pBlueScript KS between KpnI and EcoRI, T3 transcription,
termination by NcoI digestion with the plasmid) with either 5'-CAP
(1; 4), 5'-phosphate (2; 5) or 5' --OH (3; 6) termini generated by
standard methods.
1 10 .mu.l (8 ng/.mu.l) RNA [89 ng] 1 .mu.l (1 .mu.M) oligo
(.sup.32P-labl.) [0.05 .mu.M each] 4 .mu.l (5x) RT buffer [50 mM
Tris-Cl 8.3, 75 mM KCl, 3 mM MgCl.sub.2] 1 .mu.l (100 mM) DTT [5
mM] 1 .mu.l (20x) MnCl.sub.2 buffer [8 mM MnCl.sub.2] 1 .mu.l (10
mM) dNTPs [0.5 mM] 1 .mu.l (40 u/.mu.l) TNAse inhibitor [40 units]
1 .mu.l (200 u/.mu.l) SuperScriptII .TM. [200 units] total volume:
20 .mu.l
[0044] A .beta.-globin-specific 5'-terminal .sup.32P-labelled 20
mer was used as the oligonucleotide (GenBank V000882, nucleotide
pos. 661-680).
[0045] The reaction mixtures were denatured for 3 min at 80.degree.
C. and shock-cooled at 4.degree. C. for 3 min before adding the
enzyme. After adding the enzyme the mixtures were incubated for 60
min at 42.degree. C. Subsequently the reaction products were
separated on a 10% polyacrylamide/8M urea gel and quantified on a
PhoshorImager. A sequence that was generated with the same
oligonucleotide from a cloned fragment of a globin gene was used as
a length control. The result is shown in FIG. 2. The transferase
activity is limited to the addition of one nucleotide in the RT
buffer without addition of manganese (1-3); the reaction occurs
with a higher processivity with an mRNA template which has a 5'-CAP
terminus (1, 73%+1) compared to 5' --OH (2, 57%+1) or 5'-phosphate
(3, 22%+1). In contrast, when a buffer system containing manganese
chloride (4-6) is used, the transferase activity of the reverse
transcriptase is not only dependent on the CAP structure at the 5'
terminus of the mRNA template but it is also much more efficient.
The proportion of molecules with more than+2nt is 91%. In the case
of 5'-CAP termini (4) it is 49% for+3nt and 42% for+4nt compared
with 57% for+1nt and 39% for+3nt in the case of 5'-phosphate (5)
and 40% for+1nt, 22% for+2nt and 34%+3nt in the case of 5' --OH
termini (6).
[0046] Alternatively manganese chloride at a final concentration of
10 mM was not added until after the cDNA synthesis before the
reaction mixtures were incubated for a further 10 min. No
significant differences with regard to the efficiency of the
tailing reaction were found compared to adding manganese chloride
at the beginning of the reaction.
Example 2
[0047] 5'-CAP-dependent Terminal Transferase Activity: Comparison
of Various RTs
[0048] The experiment described in example 1 was repeated with
various reverse transcriptase enzymes. The result is shown in FIG.
3. RTs with and without RNaseH activity [Mu-MLV (3,4) and AMV (5,6)
and Expand.TM.RT (1,2) and SuperScript.TM. (7,8)] were compared
with regard to terminal transferase activity during cDNA synthesis
using a 5'-CAP mRNA template. For this the respective reaction
buffer of the manufacture was either tested in its original form
(1,3,5,7) or containing 8 mM manganese chloride as an additive
(2,4,6,8). A manganese chloride dependent transferase activity was
only found in the case of the RNaseH-deficient enzymes. In this
connection SuperScript.TM. and Expand.TM.RT only differed slightly
with regard to the efficiency of the incorporation of 3 or 4
cytosine residues (Expand.TM.RT (2): +3:43%, +4:31%;
SuperScript.TM. (8): +3:8%, +4:81%.
Example 3
[0049] Terminal Transferase Activity of the Expand.TM.RT: Manganese
Dependency
[0050] The experiment described in Example 1 was repeated as shown
in FIG. 4 with the Expand reverse transcriptase.TM. (Roche
Molecular Biochemicals) and various manganese chloride
concentrations (0 mM (1), 2 mM (2), 4 mM (3), 8 mM (4), 10 mM in an
additional 15 min incubation after the cDNA synthesis (5)). The
following efficiencies with regard to the incorporation of a 3rd
and 4th nucleotide were determined: 23% (0 mM), 43% (2 mM), 63% (4
mM), 73% (8 mM), 70% (10 mM, post incubation).
Example 4
[0051] rATP-tailing with Terminal Transferase (ribo-tailing
[0052] After a suitable purification (Roche Molecular Biochemicals
High pure Purification Kit) the cDNA synthesized according to the
invention from example 1 was incubated in the following reaction
mixture for 30 min at 37.degree. C.:
2 20 .mu.l cDNA 6 .mu.l (5 x) TdT buffer [200 mM K-cacodylate, 25
mM Tris-Cl 6.6, 0.25 mg/ml BSA] 3 .mu.l (25 mM) CoCl.sub.2 [2.5 mM]
0.5 .mu.l (10 mM) ATP [167 .mu.M] 0.5 .mu.l (25 U/.mu.l) terminal
[12.5 units] transferase total volume: 30 .mu.l
[0053] In order to analyse the efficiency of the reaction the
experiment was repeated with a .sup.32P-labelled
deoxy-oligo-nucleotide (20 mer) instead of cDNA under the same
reaction conditions. The result is shown in FIG. 5: The 30 reaction
products were separated on a 20% polyacrylamide/8 M urea gel and
quantified on a PhosphorImager. A total reaction efficiency of 94%
was determined with regard to the addition of 3 (64%), 4 (25%) or 5
(5%) adenosine residues.
Example 5
[0054] Selective Ligation to the Adaptor
[0055] After a standard ethanol precipitation the cDNA which was
previously treated with terminal transferase was resuspended in 15
.mu.l water and ligated with a population of adaptor molecules: The
adaptors were composed of a double-stranded region of 50 base pairs
with a 6-7 nucleotide 3' overhang of [5'-T.sub.3-4G.sub.3-3']. The
ligation mixture was composed as follows:
3 13 .mu.l ATP-tailed cDNA 2 .mu.l (50 ng/.mu.l) 56/57 bp adaptor
[100 ng, 3 pmol] 2 .mu.l (10 x) ligation buffer [66 mM Tris-Cl 7.5,
5 mM MgCl.sub.2, 1 mM DTT, 1 mM ATP] 1.5 .mu.l DMSO [7.5 vol %] 1.5
.mu.l (1 u/.mu.l) T4 DNA ligase [1.5 units] total volume: 20
.mu.l
[0056] After incubation for 5 min at 37.degree. C. and 2 min at
24.degree. C. to selectively hybridize the adaptor overhang to the
3'-tailed end of the cDNA, the mixture was incubated for 16 h at
16.degree. C. for ligation.
[0057] The selectivity of the ligation to the adaptor was also
tested in an assay using .sup.32P-labelled oligonucleotide (20
mer). The result is shown in FIG. 6. Oligonucleotides with
compatible (3'-CCC, 1-4) and incompatible (3'-AGC, 5-8) 3'-termini
were compared. The oligonucleotides (1 or 5) were ligated to the
adaptor [5'-G.sub.3T.sub.3-4] after rATP tailing as described in
example 4 (1 or 6) in a standard ligation mixture containing T4 DNA
ligase (3 or 7) or DMSO (7.5% v/v) as an additive (4 or 8). The
reaction products were separated in a 15% polyacrylamide/8 M urea
gel and quantified on a PhosphorImager. The result of adding DMSO
(4) is that 93% (compared to 77% under standard conditions, 3) of
the cDNA fraction is ligated with compatible 3'-termini.
Furthermore the ligation conditions are selective i.e. even
molecules which are terminated by a C are only ligated under these
conditions by 14% (7) or 8% (8) using DMSO in the buffer. This
results in an increase in the selective isolation of complete
cDNAs.
Example 6
[0058] Amplification of a Human GAPDH-cDNA Produced and Modified
According to the Invention Compared to Amplification by 5'-RACE
[0059] Starting with various amounts of a poly-A+ mRNA (10 ng, 2.5
ng, 0.5 ng, 100 pg, 20 pg) isolated from the human cell line MOLT-3
(ATCC strain collection No. CRL-1552), a cDNA synthesis according
to the invention was carried out according to examples 1, 4 and 5
using deoxy-cytosine tailing, a terminal transferase reaction with
rATP and a ligation with an adaptor according to the invention.
[0060] Subsequently a PCR reaction was carried out under the
following conditions for the specific amplification of GAPDH:
4 2 .mu.l aliquot of the ligation from example 5 1 .mu.l (50 .mu.M)
primer STI-2 [1 .mu.M] 1 .mu.l (50 .mu.M) primer GAPDH-2/M [1
.mu.M] 1 .mu.l (10 mM) dNTPs [0.5 mM] 5 .mu.l (10x) Expand .TM.
buffer [Roche Molecular Biochemicals, contain- ing 1.5 mM
Mg.sup.++] 39.5 .mu.l H.sub.2O 0.5 .mu.l (3.5 u/.mu.l) Expand .TM.
[Roche Molecular Biochemicals] total volume: 50 .mu.l thermocycles:
1 x 30 sec 72.degree. C. 1 x 3 min 94.degree. C. 35 x 20 sec
94.degree. C. 30 sec 64.degree. C. 40 sec 72.degree. C. 1 x 5 min
72.degree. C.
[0061] GAPDH-2M is a 24 mer corresponding to nucleotide position
83-106 from exon 4 (GenBank acc. No. J04038) of the GAPDH gene with
a calculated melting point Tm of ca. 70.degree. C. The STI-2 primer
had a length of 20 deoxy nucleotides and a calculated melting point
Tm of ca. 69.degree. C. GAPDH was amplified in parallel mixtures
using the same amplification primers and the amplification protocol
of the 5' RACE method known from the prior art (Frohmann, M. (1994)
PCR Methods and Applications, 4,540-558).
[0062] The PCR products were separated in a 1.25% agarose gel and
evaluated by densitometry. The result shown in FIG. 7 demonstrates
that the sensitivity of the method according to the invention is
considerably higher than the 5' RACE method of the prior art.
Example 7
[0063] Direct Sequencing of the PCR Amplificate by Enhanced
CRTC
[0064] The amplification product obtained according to the
invention from example 6 was sequenced according to the principle
of the dideoxy method as described in Schmitt and Muller, Nucl.
Acids Res. 24, 1789-1791 (1996) using an IRD800
fluorescent-labelled GAPDH primer corresponding to nucleotide
position 78 to 97 from exon 3 of the GAPDH gene (GenBank acc. No.
J04038, Ercolani L., Florence B., Denaro M., Alexander M.;
Isolation and complete sequence of a functional human
glyceraldehyde-3-phosphate dehydrogenase gene; J. Biol. Chem. 263:
15335-15341 (1988)) in the direction of the transcription start. As
shown in FIG. 8 the sequence allows the exact allocation of the
transcription start (arrow) of the human GAPDH gene.
[0065] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention. Various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims. All publications cited
herein are incorporated by reference in their entirety.
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