Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-16
2004-10-19
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06806049
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for analyzing expression frequencies of genes. More precisely, the present invention relates to a method that enables analysis of types and amounts of mRNA expressed from all of genes coding for proteins in a cell even with a small amount of biosample in order to elucidate dynamic change of gene expression.
BACKGROUND ART
The total number of genes on the genome coding for proteins is expected to be about 100,000 for human. As for yeast, of which total genomic structure have already been elucidated, the number of genes coding for proteins are estimated to be about 5000.
In recent years, public gene databanks have been established mainly in Europe, the United States and Japan. An enormous amount of gene information has been registered at such databanks from all over the world, and further information is newly coming together into the databanks every day. The human genome project is currently being pursued in a worldwide scale aiming at elucidating the total genes of human genome by the year of 2005, and the gene information obtained in that project is also being registered at the databanks. By inquiring to such databanks about a certain gene sequence, one can know if any gene having the same sequence as, or analogous sequence to the gene sequence has already been registered or not and, if registered, information concerning the sequence including designation and function of the gene, related references and so forth. Such a search is called homology search. There are several kinds of software for performing homology search. However, when a large number of samples must be searched for, BLAST is usually used, of which searching time is short.
Usually, all of genes contained in a cell are not necessarily transcribed into mRNA so as to produce proteins from mRNA, and it is estimated for human that about 15000 genes are expressed in one cell. Thus, in a cell, many kinds of genomic genes are expressed, and a corresponding number of types of messenger RNA (henceforth referred to as “mRNA”) are produced. However, types and amounts of expressed genes (also referred to as “genetic expression frequency information” hereinafter) may vary depending on the types and conditions of cells. For example, when a blood stem cell differentiates into a lymphocyte precursor cell, pre-B cell, B cell, and then activated B cell, each cell shows entirely different gene expression, although there are also genes that are commonly expressed in them.
Measurement of such genetic expression frequency information as described above is called genetic expression profile analysis. Substances responsible for cellular life activities are mainly proteins, and it is important to analyze types and amounts of proteins translated from mRNAs as the genetic expression analysis. However, it is technically difficult under a current situation to obtain profiles for the total proteins. On the other hand, measurement of total types of mRNA has already become possible.
The method reported for the first time as the genetic expression profile analysis method is the Body Map method (Gene, 174, 151-158 (1996)). The outline of the Body Map method is as follows. A poly(T) sequence on a vector is annealed to a poly(A) tail at the 3′ end of each mRNA, and a cDNA is synthesized by using the vector poly(T) sequence as a primer. Further, the cDNA is digested with a restriction enzyme MboI. Since one of MboI site exists in every 300 base pairs of cDNA in average, the cDNA on the vector is digested into a length of 300 base pairs in average. At this time, a cDNA fragment nearest the poly(A) tail remains ligated to the vector. The vector having this cDNA fragment is each cyclized and introduced into
Escherichia coli
to prepare a cDNA library. About 1000 clones are arbitrarily selected from the library, and the nucleotide sequence for 300 base pairs in average of each clone is determined. The clones were divided into groups of clones having the same sequence, and type and occurring frequency of each sequence are calculated to obtain genetic expression profile. Homology search (BLAST search) is performed in a databank for each cDNA sequence, and clones containing genes having the same sequence as known genes are given the names of the genes. When the sequence is not registered at the databank, it is considered that no gene corresponding to the sequence exists.
In order to perform homology search by the BLAST search, information for at least 11 base pairs is required. The types of sequences consisting of 10 nucleotides are about 1,000,000, and this number is far beyond the number of gene types of which existence is expected in human, i.e., 100,000. That is, if there is information for 11 base pairs, a gene having the sequence can be identified and thus the genetic expression profile analysis is possible. Therefore, if, aiming at increasing the efficiency of the genetic expression profile analysis by Body Map which requires much sequencing, cDNA fragments of about 300 base pairs used in Body Map are further made into short fragments of 11 base pairs or more (called “tag”), many of these fragments are ligated and inserted into a vector to prepare a library of ligated tags, about 1000 clones are arbitrarily selected as in Body Map, and DNA sequences of the ligated tags are determined, it is expected that more genetic expression information can be obtained with the same labor compared with Body Map. Each tag represents a gene sequence, and occurring frequency of the tag indicates expression frequency of the gene. Since the length of DNA sequence that can be determined by once of sequencing is usually about 600 base pairs, DNA sequences of about 50 tags at most can be determined by once of sequencing. That is, it becomes possible to perform the genetic expression profile analysis with efficiency about 50 times higher than that of the Body Map method.
As a method for genetic expression profile analysis based on the aforementioned concept, there is the method of serial analysis of gene expression (SAGE, U.S. Pat. Nos. 5,695,937 and 5,866,330, European Patent Publication No. 0761822 A). In SAGE, cDNA is produced by using a poly(T) of which 3′ end is bonded with biotin as a primer, the cDNA is digested with a restriction enzyme such as MboI (called an “anchoring enzyme”) as in Body Map, cDNA fragments containing the 3′ end to which biotin is bonded are adsorbed on avidin beads, the beads are divided into two of portions, and two kinds of linkers (A or B) are each ligated to the cDNA fragments (about 13 bp) adsorbed on either of the two portions of the beads. Each linker contains a site for a Class II restriction enzyme such as BsmFI (called a “tagging enzyme”). Each cDNA fragment is excised from the beads with the tagging enzyme, the excised end is blunt-ended, and the tags ligated to the linker A and the linker B are connected. The product of the connection is called a “ditagt”. The ditag is amplified by PCR using primers that recognize the linker A and the linker B. A large number of amplified ditags are ligated, inserted into a vector, and sequenced. About 50 tag sequences can be obtained by once of sequencing. By calculation based on this tag sequence information, genetic expression frequencies are provided.
Further, as other methods for analyzing expression frequencies of genes, there are the gene chip method and the gene microarray method. In both of the methods, there are used gene fragments adhered in array to a suitable plate (usually slide glass) at an extremely high density (about 10 fragments/mm
2
or more). The gene fragments on this chip are hybridized with fluorescence-labeled mRNAs to determine types and amounts of mRNAs.
As described above, several methods have been developed for analyzing expression frequencies of genes, and fair results have been obtained. Currently, the SAGE method is the most effective means for measuring expression frequencies of the total genes of all eukaryotic organisms. However, when this method was actually practiced, it encountered many
Date Masayo
Fukuda Hisao
Maekawa Takami
Mitsui Akira
Takahara Yoshiyuki
Ajinomoto Co. Inc.
Fredman Jeffrey
Sakelaris Sally
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