Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-07
2002-01-29
Whitendant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S091510, C436S094000, C536S023100, C536S024330
Reexamination Certificate
active
06342376
ABSTRACT:
This application is a continuation of German Application No. 19840731.9, filed on Sep. 7, 1998.
1. Field of the Invention
The invention relates to a novel method for analyzing the composition of an mRNA sample and analyzing differential gene expression using two differently labeled primers, and to the use of the method.
2. Background of the Invention
Differential RNA display (DD) is one of the methods most frequently used for detecting and isolating regulated genes (Liang, P. and Pardee, A. B. (1992) Science 257, 967-971; Liang and Pardee (1995) Current Opinion in Immunology 7, 274,280; McClelland et al. (1995) Trends in Genetics 11: 242-246). The Differential Display and Reverse Transcription (DDRT) method initially involves reverse transcribing isolated RNA using a first primer in a reverse transcription (RT) reaction, thereby preparing the first strand of a complementary DNA (cDNA). In the following step, this cDNA is amplified by the polymerase chain reaction (PCR) using the first primer and a second primer. The amplified cDNA sample is then analyzed, for example, by fractionating the amplified cDNA in a gel. Detection of the PCR product can be achieved, for example, by hybridizing with a labeled probe or by labelling the amplified cDNA. Detection can also be achieved by carrying out the amplification in the presence of radioactively labeled nucleotides, usually radioactively labeled dATP, or in the presence of a labeled primer which is, for example, labeled with a fluorescence (“fluorescence DDRT’) (Ito et al. (1994) FEBS Letters 351, 231-236; Ito and Sakaki (“Methods in Molecular Biology Vol. 85: Differential Display Methods and Protocols” (1997) p. 37-44 Liang and Pardee eds., Human Press Inc. Totowa, N.J.; Jones et al. (1997) Biotechniques 22, 536-543; Smith et al. (1997) Biotechniques 23, 274-279).
If the reaction is carried out in the presence of radioactively labeled nucleotides, all the amplified cDNAs are then labeled. This technique is also known as radioactive DDRT or classical DDRT. If, on the other hand, a labeled primer is used (e.g. for fluorescence DDRT), only a part of the amplified cDNAs is labeled because of the possible primer combinations of the first labeled primer and of the second unlabeled primer during the PCR.
The first labeled primer, primer 1, is in this case, an oligo(dT) primer which possesses two further nucleotides (M=A, C, G; N=A, C, G, T) at the 3′ end. Thus “5′-(T)
12
-MN-3′” is equivalent to “(T)
12
-MN.” Labeled primer 1 is used in the RT reaction. The second unlabeled primer, primer 2, is in this case, an oligonucleotide which has a sequence of 10 randomly ordered nucleotides (“10 mer”). Labeled primer 1 and unlabeled primer 2 are used in the subsequent PCR; however, primer 1 is customarily used in a twofold excess. In this way, a relatively high proportion of amplified cDNAs whose 3′ ends exhibit the sequence of the first primer and whose 5′ ends exhibit the sequence of the second primer (e.g. 5-10 mer - - - (T)
12
-MN-3′, where “ - - - ” symbolizes the amplified cDNA sequence which is located between the primer sequences) is obtained as a rule.
The resulting PCR reaction potentially gives rise to four types of PCR products as listed below. The first product is as described above, where 3′ ends exhibit the sequence of the first primer and 5′ ends exhibit the sequence of the second primer (i.e. product 1). The second product contains primer sequences that are transposed as compared with product 1, at whose 5′ and 3′ ends can be found the sequences of the (T)
12
-MN primer and of the 10 mer primer, respectively (i.e. product 2). Other cDNAs are either amplified only using the (T)
12
-MN primer (i.e. product 3) or only using the 10 mer primer (i.e. product 4). Consequently, the use of two different primers can result in the amplification of the following reaction products in a DDRT as a result of the possible primer combinations:
Product 1) 5′-10 mer - - - (T)
12
-MN-3′
Product 2) 5′-(T)
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-MN - - - 10 mer-3′
Product 3) 5′-(T)
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-MN - - - (T)
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-MN-3′
Product 4) 5′-10 mer - - - 10 mer-3′
Owing to the fact that only the first primer is labeled in this fluorescence DDRT, only those cDNAs which were amplified in at least one direction using the labeled first primer, products 1, 2, and 3, are detected in the subsequent analysis of the amplified cDNAs. In contrast, cDNAs which are only amplified using the second primer are neither labeled nor detected (i.e. product 4). Consequently, the complexity of the detected PCR products (cDNAs) is less than that in a radioactive DDRT. For this reason, the conventional fluorescence DDRT does not detect those regulated genes, or the mRNAs which correspond to them, which are only amplified using the second primer. As a result, the number of regulated genes detected by the conventional fluorescence DDRT can be lower than that detected in a radioactive DDRT.
Furthermore, when one single mRNA is used, a uniform cDNA product is not amplified in the PCR either with conventional radioactive DDRT or with conventional fluorescence DDRT, as shown above in products 1 to 4. Instead, several cDNAs which differ in their length, as seen by fractionating in a gel, can be amplified as a result of the different primer combinations. Only a detailed analysis such as direct sequencing would show whether different amplified cDNAs are cDNA products of one and the same regulated gene or not. The conventional DDRTs do not provide the possibility of differentiating between the redundant labeled, amplified cDNAs. In a radioactive DDRT, it is not possible to differentiate between products 1 to 4. In a conventional fluorescence DDRT, it is not possible to differentiate between products 1, 2, and 3 although there is a greater probability that the visible cDNA fragment derives from the 3′ region of a gene since the 3′ primer is labeled with the fluorescence dye.
Consequently, the redundancy of amplified cDNA fragments in conventional radioactive DDRT cannot be established or can only be established with a relatively large amount of effort. On the other hand, conventional fluorescence DDRT results in a lower complexity of the amplified cDNA product as compared with conventional radioactive DDRT. Furthermore, conventional fluorescence DDRT runs the risk of overlooking particular regulated genes. These are the main problems of conventional radioactive DDRT and conventional fluorescence DDRT methods, respectively.
SUMMARY OF THE INVENTION
The invention on which the present application is based provides a method which can be used for analyzing RNA samples and in particular for analyzing differentially regulated genes. This method does not suffer from the abovementioned disadvantages.
The present invention relates to a method for analyzing an RNA sample, preferably a mRNA sample, which comprises:
a) using a first primer, which is, where appropriate, labeled with a first dye, to prepare the first strand of a complementary DNA sample (cDNA sample) from an RNA sample, preferably an mRNA sample,
b) using a second primer, which is preferably labeled with a second dye, to prepare the second strand of this cDNA sample,
c) using the first primer, which is labeled with a first dye, and the second primer, which is labeled with a second dye, to amplify the cDNA sample, and
d) analyzing the composition of the amplified and labeled cDNA sample.
The RNA sample contains mRNA to be analyzed. In a preferred embodiment of the invention, mRNA is used in the method. The RNA can be isolated, for example, by known methods such as CsCl
2
density gradient centrifugation or column chromatography. The RNA is preferably isolated from a cell or a population of cells or from tissue. Where appropriate, the mRNA can be enriched from the RNA, for example by means of chromatography through an oligo (dT) column.
The method employs a first primer and a second primer. The first primer is the primer which is used for the reverse transcripti
Kozian Detlef
Reuner Birgit
Aventis Pharma Deutschland GmbH
Finnegan Henderson Farabow Garrett & Dunner
Lu Frank
Whitendant Ethan
LandOfFree
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