Navigation Links
Serial Analysis of Gene Expression Using ABI PRISM DNA Sequencers and BigDye Terminators

Serial Analysis of Gene Expression (SAGE) is a sequence-based approach to the identification of differentially expressed genes by comparative analyses. SAGE is a powerful tool, one of the more comprehensive methods available for detailed analysis of large numbers of cellular transcripts, leading to a profile of the expressed genes.1 SAGE provides an accurate quantitative analysis of the relative levels of genes expressed in a specimen. The ability to count many thousands of genes allows the detection of genes that are expressed at very low levels in a high-throughput manner. A three-step molecular approach, the SAGE process allows simultaneous analyses of sequences derived from various cell populations or tissues.2


In 1995, Victor Velculescu first described SAGE, a pioneering approach to gene expression analysis that enabled a detailed characterization of expression patterns without prior knowledge of previously isolated genes. The method was originally developed to investigate genes that might be differentially expressed in colon cancer.4

Cancer research remains the focus of the vast majority of SAGE studies. The identification of novel markers for cancer progression and prognosis as in the case of uterine cancer, in which early stage I detection can be difficultis critical for successful treatment. The SAGE method can identify potential targets for therapeutic intervention and find key biochemical pathways. This application is well suited for gene expression analysis of any tissue from any organism.

SAGE is based on the principle that once isolated from a defined position within a transcripta sequence tag of 9 or 10 base pairs (bp) provides sufficient information to identify a transcript. These tags can be serially analyzed by ligating or concatenating them to form longer chains. After they are sequenced, software tools are used to identity and determine abundance. This process involves the following three steps:

1.Biotinylated primers are used to reverse-transcribe mRNA. The resultant cDNA is then digested with a frequent-cutting enzyme (anchoring enzyme AE). Using streptavidin-coated beads, the 3' end of the cDNA is bound and then split into two pools, A and B. A primer (A' and B') containing a recognition site for a type IIS restriction enzyme is ligated to the nucleotides in each of these pools.

2.Following digestion with a relevant type IIS tagging enzyme, eluted DNA fragments are ligated and amplified using the A' and B' primers. Following PCR, the anchoring enzyme removes the priming sites A and B. Concatamers are then formed and cloned into a vector. A ditag containing the sequence information of two independent cDNA tags is punctuated by the anchoring site.*

3.Cloned inserts are verified by agarose gel and sequenced. SAGE analysis tools then analyze these sequences. Finally, a comparison of the sequence data determines variations in expression patterns.

Applied Biosystems Capillary Electrophoresis Systems and SAGE Technology

Most SAGE investigations require analysis of more than 50,000 tags. Obtaining sequencing information quickly and accurately is vital. Applied Biosystems capillary electrophoresis systems and BigDye terminator chemistries offer an easy, hands-free approach to sequencing. ABI PRISM 3700 and 3100 DNA sequencers accurately and reliably determine the transcript information necessary to build a profile. ABI PRISM BigDye terminators provide signal uniformity and sensitivity. Together they offer a cost-effective solution to largescale transcript analysis using SAGE technology.

Signal Detection

Accurate basecalling at long readlengths is important for ditag analysis. BigDye terminators provide the hig h degree of accuracy that transcript analysis of short 9- or 10-bp sequences demands. This accuracy, coupled with the sensitive detection capacity of the ABI PRISM 3100 or 3700 DNA sequencers, ensures a fast and accurate read. BigDye Terminator v 3.0 enables even greater signal uniformity for samples that show a tendency toward signal loss in longer fragments.

Complete Automation

Both the 3100 and 3700 DNA sequencers are designed for more than 24 hours of unattended operation. Human intervention is required only to place the 96- or 384-well sample trays on the instrument, and to replenish the polymer supply. All other steps, including capillary filling, sample loading, capillary rinsing, data extraction, and analysis are completely automated.

SAGE Sequence Protocol

In experiments using typical sequencing methods, along with the ABI PRISM 3100 Genetic Analyzer and 3700 DNA Analyzer for sequencing, Applied Biosystems scientists produced reproducible and accurate ditag sequences.

Samples were prepared from mRNA derived from a cell line, Mcf-7 (human breast adenocarcinoma). A sequence content of approximately 26 ditags was obtained using both the standard protocol with POP-5 polymer on the 3700 DNA Analyzer, and the Rapid Read protocol on the 3100 Genetic Analyzer. For optimal sequence efficiency, the insert length should be sufficient to fully utilize the length-of-read for the instrument platform and BigDye chemistry.


Throughput is an important consideration when choosing a platform for sequence analysis. The elimination of gel tracking and other problems that can arise from slab-gel sequencing greatly improves the sequencing capacity of the average research laboratory. Throughput requirements depend on the quantity of tags to be analyzed. ABI PRISM DNA sequencers provide this flexibil ity by offering medium-to-high throughput.

Capillary lengths of 36 cm, 50 cm, and 80 cm on the 3100 Genetic Analyzer afford flexible run times and read lengths. This system is designed with 16 capillaries, and run times can be determined, depending on insert size. Typically, a rapid run will read tag sequences to 550 bp with 98.5% accuracy. We recommend the 80-cm array for long-read runs for inserts of 1 kb or larger. (See Table 1 for throughput.)

Using standard run conditions, the 50-cm array on the ABI PRISM 3700 DNA analyzer will provide long reads with POP-5 polymer over a 3-hour run time. Three 24-hour running periods, therefore, will yield almost all the sequence information required to profile one tissue type (See Table 1).


When sequencing and basecalling are complete, the investigator needs to assess the presence and frequency of ditags. Various software tools are available to analyze the ditag sequence and determine transcript abundance. When all sequence files have been processed, it is possible to view tag abundances, match tags to reference sequences, and compare tag abundances from multiple projects. Analysis generally involves matching the tag information against the GenBank-UniGene5 database and generating a reference list. Project tags are compared with this list to identify matches of known genes and other sequences.

The National Institutes of Health (NIH) has established SAGEmap, a public repository and resource for SAGE data.6 Originally developed by researchers at the National Center for Biotechnology Information (NCBI) in Bethesda, MD, SAGEmap was designed to archive SAGE data produced through the Cancer Genome Anatomy Project (CGAP). However, it also accepts submissions of SAGE sequence from any source. The National Cancer Institute (NCI) has chosen to use SAGE exclusively for The Human Tumor Gene Index (hTGI) initiative, which is part of CGAP.


SAGE is a well-recognized method of gene-expression profiling. The information gathered from SAGE analysis enables a clear view of the genes associated with normal, developmental, and disease states. Researchers require an efficient means of accessing the sequence information contained in the concatamer of transcript tags. ABI PRISM 3100 and 3700 DNA sequencers, coupled with BigDye terminator chemistry, provide a versatile approach to generating ditag sequences, and make the SAGE application readily accessible to disease investigators.


1.Velculescu, V.E., Zhang, L., Vogelstein, B., and Kinzler, K.W. 1995. Serial analysis of gene expression. Science . 270:484487.

2.Kozian, D.H., and Kirschbaum, B.J. 1999. Comparative gene-expression analysis. Trends in Biotechnology . 17:7378.

3.Johns Hopkins University SAGE Web site: Schematic of SAGE method. April 2001.

4.Dr. K. Kinzler, Dr. B. Vogelstein, Johns Hopkins University, Baltimore, MD.

5.National Institutes of Health Genetic Sequence Database: Genbank/ and http://www.ncbi.nlm. April 2001. 6.Lash, A.E., Riggins, G.J. et al. 2000. SAGEmap: A public gene expression resource. Genome Research. 10:10501060.

*Invitrogen Corporation has released I-SAGE, the first pre-assembled kit for performing SAGE technology. This all-inclusive kit provides sufficient pre-tested materials to generate up to six SAGE libraries for subsequent sequencing.



Page: All 1 2 3 4 5

Related biology technology :

1. Serial Dilution with the Bravo Liquid Handling Platform
2. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
3. Epitope-Tagging Vectors for Functional Analysis in Yeast
4. Map and Link Human Genetic Disorders with SSLP Analysis
5. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
6. Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf cMaster RTplusPCR System
7. Structural Analysis of Glycosylated Peptides in Complex Mixtures with Ion Trap MSn
8. Use of Transcriptor Reverse Transcriptase in Microarray Analysis
9. Increased Sensitivity in Microarray Analysis
10. Gene-Expression Analysis of TP, DPD, and TS Using the LightCycler mRNA Quantification KitsPLUS
11. Agilent 2100 Bioanalyzer Automated Analysis System
Post Your Comments:

(Date:10/12/2017)... ... October 12, 2017 , ... They call it the “hairy ... a depiction of a system of linkages and connections so complex and dense ... of computer science at Worcester Polytechnic Institute (WPI) and director of the university’s ...
(Date:10/12/2017)... FL (PRWEB) , ... October ... ... (RPS®) today announces publication of a United States multicenter, prospective clinical study ... use, disposable, point-of-care diagnostic test capable of identifying clinically significant acute bacterial ...
(Date:10/12/2017)... ... October 12, 2017 , ... ... Rosalind™, the first-ever genomics analysis platform specifically designed for life science researchers ... honor of pioneering researcher Rosalind Franklin, who made a major contribution to ...
(Date:10/11/2017)... NJ (PRWEB) , ... October 11, 2017 , ... Personal eye wash is a basic ... one eye at a time. So which eye do you rinse first if a dangerous ... have Plum Duo Eye Wash with its unique dual eye piece. , “Whether ...
Breaking Biology Technology:
(Date:4/24/2017)... , April 24, 2017 Janice ... partner with  Identity Strategy Partners, LLP (IdSP) , ... or without President Trump,s March 6, 2017 ... Entry , refugee vetting can be instilled with greater ... (Right now, all refugee applications are suspended by ...
(Date:4/17/2017)... NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or the "Company"), ... Report on Form 10-K on Thursday April 13, 2017 with the ... The ... section of the Company,s website at  under "SEC Filings," ... 2016 Year Highlights: Acquisition of ...
(Date:4/11/2017)... PALM BEACH GARDENS, Fla. , April 11, ... biometric identity management and secure authentication solutions, today ... million contract by Intelligence Advanced Research Projects Activity ... technologies for IARPA,s Thor program. "Innovation ... the onset and IARPA,s Thor program will allow ...
Breaking Biology News(10 mins):