Fragment Analysis Information
Our facility has a service to carry out fluorescent DNA fragment analysis--this can include analyzing labeled products generated by AFLP, STR, or even DNAse footprinting or primer extension. We have the most experience using the Genemarker software package from SoftGenetics, run on capillary instrumentation. Because of the different types of experimental manipulations and output that fall under the heading "fragment analysis," we have numerous options for sample preparation, drop off, and computer analysis.
Generation of labeled fragments
All the PCR reactions are run in your lab--we don't do that here in the facility. New users are encouraged to run a sample of their reaction products on an agarose gel to insure that something is there before bringing it to our facility. Many of the amplification procedures are tricky.
Fluorophores:
Our ABI instrumentation is spectrally calibrated to detect a number of fluorophores. On the 3730, our highest output machine (12-16 96 well plates/day, meaning shorter turnaround times in general for dropped off samples) we can run filter set D and filter set G5 samples. These include the dyes 6-FAM (blue), HEX (green), NED or TAMRA (yellow), and ROX (red) for filter set D. ROX is reserved for the internal MW standard run in every sample on filter set D. FAM, TAMRA and HEX are commercially available dyes, meaning we can synthesize primers containing these dyes in house. NED primers are only available from Applied Biosystems/Life Technologies. The G5 filter set allows for four dyes attached to samples, using LIZ as the internal lane standard. The dyes used for samples in G5 are: FAM, HEX, NED or Tamra, and PET. PET is only available from Applied Biosystems/Life Technologies.
It is unwise to go back and forth between instruments too, unless each series of runs represents an independent experiment. For each instrument the internal consistency is excellent--run to run will give highly reproducible data. However, mobility differences between instruments are to be expected so sticking with one machine is recommended.
Sample Preparation
Following PCR, 3 ul of the amplified products are mixed with 10ul of formamide and 0.3ul MW standard, then heat denaturated and loaded onto the machine. A critical fact to note is that the samples for all the machines are loaded onto the capillaries using electrophoretic injection, meaning an electric field is applied to bring the sample to the capillary. This method is very sensitive to components in the sample, particularly salts/buffers of any kind, excess primer, or other abundant contaminants such as primer dimers or very short PCR products. Such unwanted components can inhibit injection of your sample and/or the size marker, resulting in non-analyzable data and a wasted sample. From what we see with most users, 3 ul straight out of an amplification rarely gives good data. There are 2 ways to get around this problem. The simplest is to dilute the PCR reaction in water then add diluted sample to the formamide. We recommend trying a couple of dilutions at least--1:5 and 1:20 are good places to start. The other method is to do a magnetic bead or column cleanup of the PCR products following amplification. While we have done magnetic bead cleanup of PCR products in the past, we are no longer offering that service nor do we recommend it due to potential loss of fragments. WE WILL NOT ACCEPT RESPONSIBILITY FOR POOR RESULTS ON UNCLEANED/UNDILUTED SAMPLES. Also DO NOT HAVE PVP in samples.
Another critical factor is the formamide used for dilution- NOT ALL COMMERCIAL FORMAMIDES WILL WORK! We use the ABI Hi-Dye formamide; we know that deionizedformamide from Sigma also works. If you want to use another brand of formamide, contact us first.
Our primary goal is to generate good data for our users and we want to make every effort to insure that samples look good. Therefore, for users just beginning their projects, we can run a number of samples for free so you can test cleanup and/or dilution parameters. The first step (after reading this of course) is to talk with us about what you have, and we'll suggest what to do next.
Sample Drop Off
OK, you now know how to get good amplification, how to treat your samples and you want to drop stuff off for us to process. It all happens on the sequencing drop off computer. If necessary, we'll log you in as a new user (takes about a minute) so when you come over you'll be recognized by the computer. There are various options you can select for processing of GeneScan samples.
1. If you have cleaned up your samples, or diluted them appropriately, select "Genescan Plate" and just type the name of the plate on one line only. Any sample names you type on this original sample sheet can't be used, so don't waste your time. If you want particular sample names to be used, you will need to upload an Excel file containing the names to our server--we can show you how to do this if necessary. The default names will just be well positions (A1, B1, etc). Pricing and various discounts will be applied automatically. Pricing is as follows:
- 96 samples: $96
On your order sheet, we also need you to write out the following info:
a) Which dyes are used to label your samples (this is VERY IMPORTANT).
b) The maximum fragment size expected or required. Analysis is commonly limited to fragments of about 600 bp or below, but we can go higher upon consultation. This is due to both labeled standard and software limitations. We currently keep 2 size standards on hand: ABI’s GS-120 (LIZ labeled), and Chimerx’s GeneFlo-625 (ROX labeled). For most samples the GeneFlo-625 is the best option, but if you are unsure we can help you decide which is best for your samples.
Additional sample submission guidelines:
a) Samples must be in striptubes or 96 well format. No 0.5 or 1.5 ml tubes.
b) We do not accept samples covered with oil. If that’s how you did your PCR, you’ll have to transfer them or remove all the oil.
c) Don’t dry down your samples.
d) We strongly recommend you include at least one sample representing a negative control and one well representing a positive control. That will help insure that we've loaded your samples correctly, and that you're getting what you think you're getting.
e) While we load only 3ul of sample, we want at least 5 ul to insure proper pipetting of material. It also allows us to do a rapid rerun if necessary
2. An inexpensive way to carry out fragment analysis is using the "Ready-To-Go", or RTG, option. To take advantage of this, you will mix your appropriately cleaned or diluted PCR samples with formamide and MW standard in your lab in the correct plate or strip tube format. We can provide you with the plate to use, for striptubes you'll provide your own. Cover the samples with foil and bring them over here, then select one of the option:
RTG GeneScan
1 plate $65
Data Output
Here's where it gets interesting! In our opinion, and of many in the fragment analysis community, analysis software options are limiting. It's also a rapidly changing environment, so what's true today may not be a year from now as options increase. Currently the data generated from our Applied Biosystems 3730 are in PC format. There are several options to help you analyze your data.
a) You can buy your own copy of GeneMarker from SoftGenetics (Windows only) to open the files. ABI has phased out GeneScan in favor of another program they've developed called GeneMapper. However, GeneMapper from ABI is a very expensive program ($10K or so), so we opted for GeneMarker.
b) You can buy a copy of a program called Dax for about 3,500. It is a complicated program, and it was designed to work with all kinds of chromatogram generating systems such as HPLC or GC in addition to output from our capillary sequencers. It therefore can be a bit intimidating at first, but it's very powerful and has some great features for AFLP analysis. For example, it can automatically bin the fragment data, create a graphical "binning map" and can output the data in a format compatible with phylogenetic analysis programs such as PAUP and PHYLIP. You can get more information from http://www.dax.nl/, the author is on this list if you have questions and he's very helpful. If you're intrigued we highly recommend that people at least try the time limited demo to see the power of this program. We have a copy of this program ourselves as well that you can play with and see if it's right for you.
c) You can download a FREE (PC based) program from ABI, Peak Scanner, to look at your fragments. The link is on our web page. This is a simple program with few bells and whistles.
d) We carry out analysis or perform certain data output functions for you. We have copies of GeneMarker and GeneMapper in our facility. We can work with you to generate output that serves your purposes. The options include, but are not limited to, tab delimited files (e.g. Excel readable) containing fragment sizes, or binary binning output matrices suitable for downloading into other genetic analysis programs. Charges for this will be based on standard consulting hourly rates of $60/hour (best estimates are a 2-3 hour set up time initially, then 15-30 minutes for each plate after that).