Fragment Analysis

The Applied BioSystems 3730 DNA Analyzer has the ability to determine the size of DNA fragments by using a fluorescence-based detection system, thus dispensing with the need for radioactivity or laborious manual polyacrylamide gel techniques.

As the DNA migrates through the detection cell, the 48 capillaries are simultaneously illuminated from both sides of the array by an argon-ion laser. To accomplish this, the beam from a single laser source is split using a series of mirrors to form a dual pathway. The fluorescent emissions are spectrally separated by a spectrograph and focused onto a charged couple device, which are then converted to digital information that is processed by the collection software.

A fluorescent size standard in each capillary eliminates variability. The use of five fluorescent dyes allows multiplexing of numerous fragments or poolplexing of numerous samples, thereby increasing throughput. The Genomics Facility runs a G5 filter set, enabling the visualization of fragments labeled with 6FAM (blue), VIC (green), NED (yellow), PET (red) and LIZ (orange) dyes.

GeneMapper™ software v3.7 provides a series of automatic fragment sizing, allele scoring, bin-building and auto-panelizer algorithms to assure allele calls are highly accurate. GeneMapper is available for customer use in the Genomics facility. Results are provided as .fsa files and can be viewed by GeneMapper as well as other genetic analysis programs. Results can also be prepared as spreadsheets in an Excel tab delimited file. Parameters for this report need to be client customized.

Fragment analysis samples should be provided in 96-well format.

AFLPs (Amplied Fragment Length Polymorphism)

AFLP is a DNA fingerprinting technique which detects multiple DNA restriction fragments by means of PCR amplification. The AFLP® technology was developed by Keygene in the early 1990's and has since become one of the most popular genetic fingerprinting technologies world wide.

AFLP technology is PCR-based and requires only minimal amounts of starting DNA.

• AFLP markers have proven to be robust, reliable and reproducible.
• AFLP analysis requires no prior sequence knowledge of the target genome.

Microsatellites

Microsatellites are stretches of DNA that consist of tandem repeats of 1-6 nucleotides and are flanked by sections of conserved DNA. Repeats of one base are referred to as mononucleotides, repeats of two bases are called dinucleotides and similarly trinucleotides, tetranucleotides and pentanucleotides are repeats of 3, 4 and 5 bases. The conserved flanking regions on each side of the repeat offer an area for primer design and permit amplification of the microsatellite by PCR.

Analysis of Fragment Banding Patterns

DNA fingerprints are banding patterns of DNA fragments that are generated in various ways and then separated by electrophoresis. These differences are characteristic and heritable. With AFLP studies, the bands are generated by restriction digests of the genomic template and the subsequent ligation of adaptor oligonucleotides. A subset of all the fragments is then amplified with preselective fluorescent primers. Electrophoretic separation of the fragments reveals differences in the DNA fingerprint which are observed as the presence or absence of bands. The differences in fragment lengths can be traced to base changes in the restriction site or the primer extension site, or to insertions or deletions in the body of the DNA fragment.

With microsatellites, the number of nucleotide repeats determines the size of the allele and hence determines the banding pattern.

References

ABI Prism® 3100 Genetic Analyzer System Profile Publication 106MI18-01 Applied Biosystems 3730/3730xl DNA Analyzer User Guide for use with Data Collection Software v2.0 P/N 4347118 Rev B

ABI Prism® GeneMapper™ High Peformance Genotyping Software for Microsatellite- and SNP-Based Genotyping Applications Publication 107PB03-03.

AFLP™ Plant Mapping Kit The PCR Marker of Choice for Plant Mapping

Perkin-Elmer Corp Publication 775601-001