Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-09
2004-03-16
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091520, C536S024320, C536S024330, C536S025320
Reexamination Certificate
active
06706476
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an improved method for producing amplified heterogeneous populations of cDNA from limited quantities of RNA or other nucleic acids.
BACKGROUND OF THE INVENTION
Selective amplification of cDNAs represents a major research goal for molecular biology, with particular importance in diagnostic and forensic applications, as well as in general manipulation of genetic material.
In many important areas of research, such as in studying gene regulation in complex biological systems (e.g., the brain) having multiple phenotypes, the obtaining of an sufficient amount of RNA for isolating, cloning, and characterizing of specific regulated transcripts is problematic. Research has been hindered by, e.g., the high complexity of the mRNA, the relatively low abundance of many expressed messages, and the spatially limited expression of these messages. In particular, the isolation of sufficient RNA for micro-array analysis has been a challenge. Various labeling techniques have been developed for that purpose. These technologies can be divided into PCR and non-PCR based labeling technologies. Two of the non-PCR based methods are from NEN life science and Genisphere respectively and are based on a principle in which the detectable signal is amplified after the final hybridization.
For instance, NEN life science has developed a technology which is based on their Tyramide Signal Amplification™ (TSA) system, U.S. Pat. No. 5,196,306, which was originally developed for immunohistochemistry but has recently been adapted for micro-array analysis. Furthermore, the company Genisphere has developed their so called “Dendrimer technology” which is based on a labeled complex DNA structure which hybridizes to a target sequence. Both of these technologies have very complex protocols which require additional steps after the final hybridization of the probe to the target sequence.
Affymetrix has developed a method where the RNA is amplified prior to the labelling of the probe. This is accomplished by including a T7 promoter region into the oligo(dT) primer and using T7 RNA polymerase to generate multiple RNA copies by reverse transcribing the double stranded cDNA, U.S. Pat. No. 5,545,522.
In order to simplify the method and to be able to detect RNA at such low levels as in the sub-microgram range, the use of PCR in the amplification of the probe signal is needed. The polymerase chain reaction (PCR) is an extremely powerful technique for amplifying a specific nucleic acid. The use of PCR in gel based gene profiling technologies was introduced several years ago with the invention of the Differential Display technologies and their like (Liang & Pardee, 1992). Recently, a differential display approach has been developed to synthesize very sensitive micro-array probes (Trenkle et al., 1999). This method, however, is limited as it only amplifies about 5% of the RNA population at a time and furthermore is susceptible to the same problems of reproducibility as seen in the differential display method.
One of the demands of PCR is typically that the 5′ terminus and 3′ terminus sequence information is known for the synthesis of the primers. Homopolymeric tailing of the 3′ terminus (Frohman et al., 1988) and the synthesis of highly degenerate nucleotide primers (Gould et al., 1989) have been implemented to improve the range of cDNAs that can be amplified and cloned with PCR.
A number of techniques have been developed to add a sequence tag to the 5′-end of single stranded cDNA. For instance, the so called ‘Ligation-anchored PCR’ (Troutt et al., 1992) was developed in 1992 for that purpose. It makes use of T4 RNA ligase for the ligation of a single stranded oligo to the 5′-end of a single stranded cDNA template. The use of T4 RNA ligase in ligation of single stranded templates has however never been widely used due to the low efficiency of single stranded ligations compared to ligations using e.g. T4 DNA ligase. In another example the widely used PCR synthesis kit (Clontech laboratories) is adapted from a technique developed by Chenchik et al. (U.S. Pat. No. 5,962,277). This technique makes use of the terminal transferase activity of the MMLV reverse transcriptase. By use of a special oligonucleotide it is possible for the reverse transcriptase to switch template and extend the first strand synthesis into a specific sequence. However, only full length cDNAs will be tailed with the specific sequence anchor and only sequences which have been tailed by terminal transferase activity of the reverse transcriptase will be extended. In a more recent article, the ligation of a fluorescent labeled primer toward an unlabeled 5′-phosporylated primer was efficiently carried out using T4 DNA ligase and a so called bridging primer (Jang & Steffens, 1997). This method describes the ligation of small fluorescently labeled oligonucleotides with known sequences but is not applicable for adding sequence tags to the 5′-end of e.g. cDNAs of unknown sequences.
In spite of such recent advances, including PCR and its various modifications noted above, there is a need for irmproved methods for amplifying RNA for cloning and micro-array experiments. Especially a method is sought for that is simple and reproducible and that is able to amplify limited amount of cDNA starting from heterogeneous populations of RNA.
SUMMARY OF THE INVENTION
The present invention provides novel processes for nucleic acid amplification, especially suitable for amplification of low abundant cDNA originating from a source only containing the mRNA of interest in a very limited amount.
The present invention describes a method in which a specially designed adaptor with a non-specific overhang can be efficiently ligated directly to a single stranded cDNA and amplified directly in a subsequent PCR reaction. This method is especially suitable for generating labeled probes to be used in micro array hybridization experiments when only limited amounts of RNA is available.
The overall methodologies will be capable of amplifying a broad range of messenger RNAs without prior cloning into vectors and in some instances without knowledge of the sequence. This is achieved by performing a first strand synthesis of cDNA from mRNA, using a cDNA synthesis primer containing a polythymidylate region and an anchor region, said anchor region having a pre-defined nucleotide sequence (COM
1
) complementary to an amplification primer (primer #2). The pre-defined nucleotide sequence can be any desired nucleotide sequence, e.g., gene of interest or portion thereof of interest or gene fragment of interest.
Hereafter, a specially designed adaptor fragment is ligated to the first strand cDNA. Said adaptor is easily ligated to the cDNA via a non-specific overhang comprising non selective bases like deoxyinosines, which will keep the annealing temperature of the adaptor constant and also ensure a high equilibrium toward the specific single cDNA during the ligation. Furthermore, the adaptor contains a pre-defined nucleotide sequence (COM
3
) complementary to an amplification primer (primer #1). The single stranded cDNA product with ligated adaptor can hereafter be subjected to standard nucleic acid amplification procedures, e.g. PCR, using two primers (primer #1 and primer #2) which are preferably single stranded nucleotides of sufficient length to act on the cDNA template for synthesis of extension products under suitable conditions.
This method of nucleic acid amplification can for example be applied in a process for detecting expression of a gene in a pre-selected cell population wherein mRNA from said cell population is amplified according to the invention thereby determining the presence or absence of mRNA corresponding to the gene of interest or portion thereof of interest or gene fragment of interest. The cell population may e.g. be from a human tissue samples, such as from brain tissue. The cell population may e.g., be from an embryonic or fetal tissue. The cell population may be only a single ce
Pettersson Niels Bo
Thirstrup Kenneth
Warthoe Peter
Azign Bioscience A/S
Fredman Jeffrey
Frommer Lawrence & Haug
Kowalski Thomas J.
Wilder Cynthia
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