Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-05
2003-05-06
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S006120, C536S023100, C536S024200, C536S024300
Reexamination Certificate
active
06558927
ABSTRACT:
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
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
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.
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.
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.
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.
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 3′ 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
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.
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
2+
ions as well as in the presence of manganese
2+
ions. The manganese concentration to be used is preferably 1-20 mM and under optimized conditions 8 mM.
In one embodiment the manganese
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
2+
ions can be carried out directly after a first strand cDNA synthesis carried out in the absence of manganese
2+
ions in which case the reverse transcriptase used originally is still active under these conditions.
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.
In both embodiments the deoxy-nucleotide tailing in the presence of Mn
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.
Any reverse transcriptase enzymes can be used with the only restriction that the enzyme that is used should not have any RNAseH activity.
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.
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.
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.
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.
The additional double-stranded nucl
Mueller Manfred W.
Schmidt Wolfgang M.
Doyle Charles M.
Horlick Kenneth R.
Jen George C.
Pennie & Edmonds LLP
Roche Diagnostics Corporation
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