Email bio-eu@licor.com
4300 IMAGER
Applications for the 4300 Analysis System
cDNA AFLP®
Expression Analysis Methods
"Biological responses and developmental processes are precisely controlled at the level of gene expression. Information on the temporal and spatial regulation of gene expression often sheds light on the potential function of a particular gene. Hence, an essential aspect of functional genomics is the transcriptome; i.e., the analysis of expression patterns of genes on a large scale.
There are currently three high-throughput techniques for large-scale monitoring of gene expression:
- Serial analysis of gene expression (SAGE);
- Hybridization-based methods;
- Gel-based RNA fingerprinting techniques such as differential display and cDNA AFLP®.
In principle, SAGE can provide quantitative data concerning gene expression, but is expensive and labor intensive when multiple sample points are to be compared.
Microarray technology is extremely powerful in generating a broad view of gene expression. Unlike cDNA arrays, oligonucleotide arrays are able to distinguish between highly homologous sequences. However, the design of oligonucleotide arrays requires comprehensive sequence knowledge, at present only available for a small number of organisms.
cDNA AFLP® is an inexpensive, gel-based method for analysis of gene expression patterns and can be performed in any laboratory."
Click here to download the AFLP brochure
1Qin, Ling; Prins, Pjotr;, Jones, John T.; Popeijus, Herman; Smant, Geert; Bakker, Jaap; and Helder, Johannes. (2001) GenEST, A Powerful Bi-Directional Link Between cDNA Sequence Data and Gene Expression Profiles Generated by cDNA AFLP®. Nucleic Acids Research, Vol. 29, No. 7, 1616-1622. By permission of Oxford University Press.
Advantages
AFLP® expression procedure.
Overview of cDNA AFLP Analysis
After preparation of double-stranded cDNA templates, there are seven steps to perform (Figure 1):
- Restriction Digest with TaqI and MseI to generate fragments within a specific size range.
- Adapter Ligation where known adapters are ligated to the ends of the cDNA fragments produced during restriction digestion to yield templates for amplification.
- Pre-Amplification to increase the template available for selection is performed with primers complementary to the adapter sequences.
- Selective Amplification is used to amplify subsets of the pre-amplified templates using primers containing 2 selective nucleotides at the 3’ end of the amplification primers.
- Electrophoresis of these products on a denaturing polyacrylamide gel yields distinctive patterns for analysis.
- The Odyssey® Infrared Imaging System can be utilized to extract fragments of interest.
- Re-amplify fragments using the same primers and either clone or direct sequence, depending on the application.
Each of the steps summarized above will be discussed throughout this protocol.
Advantages of AFLP® expression analysis include:
Poorly characterized genomes can be analyzed.
Multiple genes/transcripts responsible for the attribute being evaluated can be identified.
Transcripts that are either non-coding or contain no open reading frame can be analyzed.
IRDyes® are much safer than the alternative radioactive labels.
Image data can be obtained with LI-COR DNA Analyzers in several hours rather than 2-4 days as with radioactive or silver staining procedures.
The sensitivity of LI-COR DNA Analyzers and the availability of IR-labeled AFLP® expression primers reduce overall cost and eliminate labeling steps.
Expression Analysis Kits
LI-COR AFLP® Expression Analysis Kits are designed as a simple, cost-effective, and reproducible method to generate expression data using LI-COR automated DNA analyzers.
LI-COR's AFLP® Expression Analysis Kit is available with either IRDye® 800 infrared dye or IRDye® 700 infrared dye labels. Each kit is sufficient for 10 cDNA templates, with up to 64 expression profiles per template. Additional supplementary reagents for conducting genome-wide expression studies are also available.
LI-COR's AFLP® Expression Analysis Kit offers a complete solution to identifying differentially expressed genes.
Features
IRDye® infrared dyes are safer than radioactive labels.
Easier, safer, and requires less labor than developing your own kits.
Image data are obtained in several hours instead of 2-4 days.
The sensitivity of LI-COR's DNA analyzer reduces costs through lower reagent usage and the elimination of labeling steps.
When used in combination with LI-COR's Odyssey® Infrared Imaging System, transcript-derived fragments can be recovered for further characterization. The sensitivity provided by LI-COR instruments, combined with the availability of optimized kits, eliminates radioactive labeling steps.
Band Extraction
Your AFLP® workflow can be further enhanced through integration with other infrared imaging products, such as LI-COR's Odyssey® Infrared Imaging System.
Gels from the 4300 DNA Analyzer can be scanned directly on the Odyssey System for easy, accurate band extraction. Extracted bands can then be sequenced using the DNA Analyzer. AFLP® expression can be confirmed by running a Northern blot on the Odyssey System. The time and expense of running samples multiple times is eliminated.
Technical Resources
Protocols:
Webinars and Video Tutorials:
Genomics Education Matching Funds Informative Webinar - Jackie Potts
PubMed
High-Throughput AFLP® Analysis Using Infrared Dye-Labeled Primers and an Automated DNA Sequencer.
A.A. Myburg, D.L. Remington, D.M. O'Malley, R.R. Sederoff, and R.W. Whetton.
BioTechniques 2001, 30(2): 348-357.A new and versatile method for the successful conversion of AFLP® markers into simple single locus markers.
Bart Brugmans*, Ron G. M. van der Hulst, Richard G. F. Visser, Pim Lindhout and Herman J. van Eck.
Nucleic Acids Research, 2003, Vol. 31, No. 10 e55.
To see other publications on cDNA AFLP click here.
