DNA Fragment Analysis

Automated Fluorescent DNA Fragment Analaysis in the CGC

3730 and 3130xl Genetic Analyzers can be used to accurately determine the size of fluorescently labelled PCR products and other types of fluorescently labelled DNA fragments in addition to DNA Sequencing.

Fragment analysis experiments will generally use a short capillary array suitable for use with 400bp or 500bp ROX- or LIZ- labelled size standard ladders. The 3730 in the CGC is configured with a 50cm array and calibrated for the DS30 4 color dye set, which uses ROX as the size standard label. It can also be calibrated for the 5 color G5 dye set on request. The 50cm array is longer than the one typically used for fragment analysis (36cm), but use of this array allows greater flexibility in switching between sequencing runs and fragment analysis runs without swapping arrays, at the cost of somewhat longer run times. The throughput afforded by the 3 fold larger number of capillaries compared to our previous instrument (ABI 3100) more than offsets the extra run time.

Supplies/Costs

Users are expected to provide their own consumable supplies, such as their PCR mixes and primers, 96-well plates and sealing film, and fluorescent size standards.

What you can buy from the CGC:

- Flat Top flexible 96-well plates.  These can be used for PCR reactions, cycle sequence reactions, and for loading samples into the 3130xl or 3730.

- Plate sealing film, for running PCR and cycle sequencing reactions.

- ABI plate sealing septa, for loading plates in the 3130xl or 3730.

- 400HD ROX size standard.

See the CGC Store list for the details.

The standard configuration for the ABI 3730, a 50cm array running POP7 polymer, allows either fragment analysis or sequence runs to be made in one hour for $45.  Projects that use a 1000bp size standard ladder can run samples on the 3130XL. This type of run is roughly equivalent to a long read sequencing run and takes about 3 hours, for $22.

Before each run fresh polymer is pumped into all of the capillaries in the array regardless of the actual number of samples being analyzed. Therefore, it is wise to plan runs in groups of 48/16 samples for best cost efficiency. The data collection software allows users to define analysis methods for each run (of 48/16 samples) loaded in a plate, which makes sharing plates with other users possible.

User's accounts will be charged $22.00 per run for either long fragment analysis or long sequencing runs on the 3130XL, or $45.00 for runs on the 3730.  Use of the instrument will always be charged on a per-run basis because that is the basis on which costs are actually incurred. The ABI sign out sheets include the instruction that users sharing a run can divide the charge to different budget numbers in sixteenths or 48's of a run increments, but that any empty wells in a run will be divided equally between the users of the run.

Practical considerations
The ABI instruments come with a software package which allows the computer to operate the machine and collect raw data files from it, the DataCollection 3.0 package.  For fragment analysis you will be using GeneMapper 3.7 or 4.0 as the data analysis application to organize and analyze the data produced. GeneMapper is a fairly complex application with a significant learning curve. The Center has training CD's which walk the user through the program and allow the user to practice as much as they want as the "course" progresses. Taking the time to work with the training CD is highly recommended for anyone planning to do fragment analysis experiments.

When you are planning an experiment it is worth checking the fragment analysis section of the protocols archive page to see if another user may have found a better or less expensive way to do the kind of experiment you have in mind. The center posts useful protocols and user tips on that page when a user finds a method or experimental design they think is especially good. The protocol archives page also contains a listing of the fluorescent dyes which can be used for labelling primers, and the "filter sets" used by the ABI Genetic Analyzers to collect and analyze data from fragment analysis experiments. You can refer to this information when designing primers.

Fragment Analysis  -  Background

Fragment analysis is a general term used to describe genetic marker analysis experiments which rely on detection of changes in the length of a specific DNA sequence to indicate the presence or absence of a genetic marker. Marker analysis is a general genetic technique in which the sequence of the gene is not directly analyzed, but the presence of a particular allele or mutant version of the allele of the gene is inferred from the presence or absence of a linked DNA sequence which can serve as a marker for the allele. Genetic markers are usually polymorphic genetic sequences contained in or near an allele of interest, such as microsatellites or RFLPs, which allow the chromosomal alleles to be distinguished. Using a marker analysis approach, inheritance patterns within a family can be traced and a mutant allele associated with a disease can be identified by comparing the alleles of affected and unaffected individuals. Also, inheritance patterns in populations of humans, animals, plants, etc., can also be traced, making genetic marker analysis quite useful in population biology and ecology studies.

Marker analysis would be used instead of direct examination of a gene sequence for several reasons: A gene location may be known, but the gene sequence has not yet been determined and thus direct sequence comparison is not possible. If markers exist close to the gene locus, then the presence of mutant versions of the gene can be inferred through marker analysis. Marker analysis is much faster then direct gene sequencing. This is very helpful in population studies where the number of analyses needed can be very large. Marker analysis is also much cheaper then gene analysis; marker analysis can generally be performed for a few hundred dollars, while sequencing a gene can cost several thousand dollars.

Microsatellite Analysis

Microsatellites are one of the most widely used types of markers for genetic mapping, linkage analysis, and to trace inheritance patterns. Microsatellites are tandemly repeated sequences 1 to 4 nucleotides long. The number of these repeats in a given microsatellite can be highly variable, the characteristic which makes them useful as genetic markers. The majority of microsatellites occur in introns or other non-coding regions of the genome. For use as a marker, the exact number of repeats in a given microsatellite is not important. The microsatellite analysis will make use of the differences in the number of repeats between the different alleles. This variation in number of repeats affects the overall length of the microsatellite. The length of the microsatellite is determined by PCR using primers that sit just beyond both ends of the microsatellite sequence, and thus generate a DNA fragment whose length depends on the number of repeats in the microsatellite. It is these fragments which are analyzed.

If the PCR reaction is performed with fluorescent dye-labeled primers, then the PCR fragments can be analyzed on a capillary DNA sequencing machine. Compared to analyzing a DNA sequencing reaction where there are fragments of every size from, say, 20 to 800 bp in length, in a fragment analysis experiment one only has to detect a few DNA fragments so a shorter capillary can be used. The use of shorter capillaries allows the analysis to run in less time than a sequencing run takes. Still, the size resolution has to be good enough to distinguish between fragments varying in length by as few as two bases. To achieve this, a size standard “ladder” has to be run concurrently in each capillary to create a standard curve of sufficient precision. The size standard should be labeled with a different colored fluorescent dye from the fragments to be analyzed. In practice, the fragments can be labeled with several different colored dyes, which allows multiplexing of numerous fragment analyses in each capillary separation run. By taking advantage of multiplexing by fragment size and dye color, and the ability of automated fluorescent genetic analyzer machines to autoload 16 samples at a time from 96-well or even 384-well microtitre plates, high throughput marker analysis experiments can be designed.

Some examples of genotyping using 3 color multiplexed fragment analysis runs. The microsatellite data were produced by a UW graduate student as part of a population biology study on a wild mouse species found only in alpine meadows.

AFLP Analysis

AFLP. or amplified fragment polymorphism, is a widely used genetic fingerprinting technique because it produces a large number of bands and markers per fingerprint and does not require any knowledge of DNA sequences in the target organism. A good basic explanation of the technique can be found on the Keygene web page.

OLA Analysis for SnPs

The oligo ligation assay combines PCR replication of a DNA region known to contain a single base pair polymorphism with an assay based on ligation, which requires a perfect sequence match between a pair of oligos and the target sequence in the amplified fragment to give a band.  If the polymorphic base is present at the point where the two oligos touch, the oligos can be ligated together and produce a band of the expected size.  The assay can be desinged to give various different sized bands by using several set of oligo probes, and different fluorescent tag colors can also be incorporated.  This flexibility allows for highly multiplexed assays to be developed.

This assay requires prior knowledge of the target DNA sequence. Alternative assays for single nulceotide polymorphisms can be done in the CGC using the multifunction plate reader or the real time PCR instrument.

SnPlex Analysis

SnPlex assays are a variation of the oligo ligation assay developed by Applied Biosystems, Inc. These assays are designed to work under standard conditions and allow a very high degree of multiplexing to maximize throughput. See the ABI web page
for more information.  There is also a .pdf file of a poster from the ABI SnPlex page which explains the assay design.