Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification.

نویسندگان

  • J J Chen
  • J D Rowley
  • S M Wang
چکیده

We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3' cDNA fragments covering hundred bases. A primer containing the 10-base SAGE tag is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with Pfu DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward the 3' end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE technique: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from multiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene expression. Second, a combined application of SAGE/GLGI can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3' cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth, a combined application of SAGE/GLGI can be applied to define the 3' boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

P-215: Discovery of A Novel APA Variant of A Human Potential Gene Based on Expressed Sequenced Tags Analysis

Background: Expressed sequence tags (ESTs) are sequences of cDNA fragments prepared from different tissue sources. There are over one million of these sequences in the publicly available database, and these sequences are believed to represent more than half of all human genes. The ESTs belong to different cDNA libraries, was prepared from one particular cell type, organ, or tumor. Therefore, th...

متن کامل

The pattern of gene expression in human CD15+ myeloid progenitor cells.

We performed a genome-wide analysis of gene expression in primary human CD15(+) myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of ...

متن کامل

Identification of novel genes expressed in Brassica napus during leaf senescence and in response to oxidative stress

Senescence is a genetically regulated oxidative process that involves a general degradation of cellular structures and enzymes and the mobilization of the products of degradation to other parts of the plant. The cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism) analysis has been used under stringent PCR conditions afforded by ligation of adapters to restriction fragments, and the use of s...

متن کامل

Radiation-induced changes in gene expression in the silkworm revealed by serial analysis of gene expression (SAGE).

Serial analysis of gene expression (SAGE) was used to examine the profile of expressed genes during embryonic development in the domesticated silkworm, Bombyx mori, after irradiation with Cobalt-60. A comparison of the SAGE sequence tags derived from irradiated embryos with those from normal embryos revealed 673 differentially expressed genes (P < 0.01 and at least three folds change). Of these...

متن کامل

Expression analyses of endoglucanase gene in Penicillium oxalicum and Trichoderma viride

The expression of endoglucanase gene and protein profile belonging to two fungal species, Penicillium oxalicum 1SMS and Trichoderma viride 156MS with high cellulase enzyme activity, was investigated. Fungal isolates were cultured on inducer CMC medium and then the amount of released sugar and protein were assayed every three days for a month, using arsenate molybdatereagent and Bradford method,...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Proceedings of the National Academy of Sciences of the United States of America

دوره 97 1  شماره 

صفحات  -

تاریخ انتشار 2000