DNA SEQUENCING

Due to the increased volume of samples being submitted for sequencing, we find it necessary to make the following changes.

1. Please label all template tube lids with the sample ID number.
2. Please label primer tube lids with the primer name.

Remember: One sequencing reaction = one template + one primer.

Also, please be sure to bring a copy of your sample submission sheet to the Core Facility when you submit your samples. We can not process samples for sequencing without a sample submission sheet.
Thanks.

Introduction to Sequencing at the Core Facility:

The Genomics Core Facility of the Heflin Center for Human Genetics offers fee based fluorescent DNA sequencing services for both UAB and external clients. The Core Facility accepts CsCl purified plasmids, mini-prep plasmids, single stranded DNA, PCR products, and BACs; the Core Facility processes customer samples according to Applied Biosystems Big Dye Terminator v3.1 Ready Reaction kit protocol. In addition to standard sequencing analysis, Factura analysis is also available. Please contact the Core Facility for more information.

Turn around time for sequencing is 2-3 business days. Samples are submitted and data retrieved through our secure server. Sequencing results are provided in the .ABI file format which can be viewed using either the Chromas or Edit View or 4Peaks programs.

For additional questions, please see our new FAQ page or contact the Core Facility.

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Fees:

Item	UAB
Individual sequencing reaction	$6/reaction
Complete 96-well plate	$470/plate
Quantitation/Dilution/Resuspension	$2/reaction

Non-UAB academic users please contact the director of the Core Facility for pricing.

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Submitting Samples:

STEP 1- CREATE A PROFILE

STEP 2-LOGIN AND SUBMIT SAMPLES

STEP 3-SUBMIT DNA

STEP 4-PICK UP RESULTS

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Template Preparation Methods:

The quality of your template greatly affects the success of the sequencing reaction. Sequencing by capillary electrophoresis is highly sensitive to sample contamination. Potential contaminants include: proteins, RNA, chromosomal DNA, non-specific PCR products, residual salts, organic chemicals (i.e. phenol, chloroform or ethanol), and residual detergents, as well as excess PCR primers, NTPs, enzyme and buffer components from PCR reactions. Poor quality DNA can cause noisy data (i.e. peaks under peaks), unusable sequence data, or weak signal in addition to diminishing the lifetime of the capillary array.

The following DNA template preparation methods have been shown to give reliable data when protocols are followed carefully. Refer to the Applied Biosystems Automated DNA Sequencing Chemistry Guide (www.appliedbiosystems.com) for more details. Please note: Other preparation methods and kits exist and these are only suggestions.

Plasmids	Qiagen, Edge BioSystems, Promega mini-prep kits***, Invitrogen PureLink Quick Miniprep Kit, Cesium Chloride centrifugation Modified Alkaline Lysis/PEG method
PCR products with NO non-specific products	USB PCR Product Pre-Sequencing Kit Qiagen Qiaquick kit Invitrogen PureLink Quick Gel Extraction Kit SAP/EXO I clean-up Exo SAP-IT - USB
PCR products with non-specific products	QiaexII Gel Extraction kit Qiaquick Gel Extraction kit
ssDNA (m13)	Qiagen Qiaprep M13 kit Thermomax procedure PEG precipitation and phenol extraction
BACs	Alkaline Lysis Cesium Chloride Qiagen Large Construct Kit

*** We have observed that DNA samples purified using spin columns often contain residual spin column resin which leads to poor sequencing results. We recommend an additional centrifugation step following spin column elution. We have found that centrifuging the eluted sample for 5 minutes at maximum speed and removing only the uppermost portion of the sample to a fresh tube avoids the carryover of the interfering column resin.

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DNA Concentration :

All samples and primers must be dissolved in distilled water and must be provided at the concentrations below. Take care in determining the concentration of your DNA as samples which are too dilute or too concentrated yield poor results.

DNA Template	Concentration	Primer concentration
PCR products : for every 100 bp fragment	1 ng/uL	1.6 uM
Plasmid: vector + insert	200 ng/uL	1.6 uM
BACs ( > 50 kb )	1 ug/uL	10 uM

• For each sequencing reaction submit 10 uL of template and 10 uL of primer (please do not mix).

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Determining DNA Quality:

The following methods should be used to examine your DNA quality:
--Agarose gel electrophoresis
-- Spectrophotometry

Agarose gels indicate the presence of non-specific PCR products and RNAs. If you find non-specific PCR products, you should use one of the agarose gel purification method listed above.

Spectrophotometry indicates the presence of protein contaminants. For spectrophotometry, the ratio of A260/A280 ration should be 1.7-1.9. Smaller ratios usually indicate the presence of proteins or organic chemicals. Ratios greater than 1.9 indicate the presence of contaminating RNA.

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Quantitating DNA :

As with DNA quality, DNA quantity is very important to the success of your sequencing reactions. Three common ways to determine DNA quantity are spectrophotometry, fluorometry, and agarose gel electrophoresis.

Spectrophotometry: In spectrophotometry you measure the absorbance (optical density or O.D.) of a sample at 260 nm. Please refer to the Applied Biosystems Automated Sequencing Chemistry Guide for more details.

Fluorometry: In fluorometry you measure the fluorescence of the dye bisBENZIMIDE (Hoechst 33258) binding to AT sequences in the minor groove of DS-DNA. This method is specific for quantitation of nanogram amounts of DNA. (See Sigma technical bulletin # MB-590; Product # DNA-QF or Picogreen from Invitrogen Cat #P-7589)

Agarose gel:  If you plan on quantitating with an agarose gel, we strongly advise using a mass ladder (VWR Scientific PN 80509-748), the mass ladder will allow a quick relatively accurate quantitation. Or using DNA Mass ladder from Invitrogen (Cat#10068-013).

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Primers:

Your decisions concerning primer sequence, method of primer synthesis, and approach to primer purification can have a significant effect on the quality of the sequencing data. Some of these recommendations are based on information that is general knowledge amongst researchers, while others are based on experience with dideoxy terminators.

1) Not all cloning vectors are created equally. Several versions of common sequencing primers exist and DO NOT hybridize well with all vectors. Check to be sure that your primer has exact homology to your cloning vector of choice.
2.) Primers should be at least 18-20 nucleotides in length to minimize the chances of encountering problems with a secondary hybridization site on the vector or insert.
3.) Primers with long runs of a single base should generally be avoided. It is especially important to avoid 3 or more G's or C's in a row.
4.) For cycle sequencing, primers with melting temperatures above 50°C generally produce better results than primers with lower melting temperatures.
5.) Primers should have a G/C content between 40 and 60 percent. For primers with a G/C content of less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the melting temperature above the recommended lower limit of 50°C.
6.) Primers should have a GC clamp on the 3' end.
7.) Primers should not contain palindromes. Palindromes can cause hairpins loops within the primer, thus resulting in an unproductive priming event.
8.) Primers should not contain sequences of nucleotides that would allow one primer molecule to anneal to itself or to the other primer used in the sequencing reactions (primer dimer formation)..
9.) If possible, run a computer search against the vector and insert DNA sequence to verify that the primer is unique.
10) Do not design degenerate primers. Do not request inosine in sequencing primers. Both degenerate primers and primers containing inosine often either fail during sequencing or give poor results.

Estimating Melting Temperature:

The following formula can be used for a rough estimate of melting temperature:

Tm = (number of A + T residues) x 2°C + (number of G + C residues) x 4°C

Primer Design Software:

There are a number of primer picking programs available for you use:

--A Mac version of Oligo 5.0 is available
--Primer 3 is a web-based primer picking program

New Services :

We have partnered with IDT to provide oligos to UAB. Please use the link to create a user profile to receive discounted oligos and free shipping. The Core will email you when the oligos have arrived, and pick them up in the Core.

We provide some free standard primers for sequencing, they are M13 forward, M13 reverse, T7 terminator, and SP6. Please check their sequences before you use them.

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Viewing electropherograms using the Chromas (PC) or EditView (MAC OS 9) or 4Peaks (MAC OS X):

Chromas (PC): To access your sequence files from the file server you will need the software program Chromas. To download the program go to: http://www.technelysium.com.au/chromas.html. A freeware version of the program can be downloaded . The latest version of the program is also available at this site for a fee.

On the left side of the website in the blue area, click Freeware Version 1.45.
Click Chromas 145-95.zip to download the program. Unzip the program and install it. If you like, create an icon on your desktop for easy program access.

Start the Chromas program by clicking the icon on your desktop. The Chromas program opens a window. On the menu bar, select 'file', then 'open', and select the sequencing results file you wish to open. The file will open to the electropherogram. To view the corresponding nucleotide sequence, first open an appropriate word processing application (Word, WordPerfect, NotePad, etc.) Next, in Chromas select 'edit' on the menu bar, then 'copy sequence'. Here you can choose to copy in either plain text or FASTA format. 'Paste' the copied sequence into the opened word processing application. 'Save' the file with containing the nucleotide sequence.

If you have any questions about Chromas, please go to official Chromas web site.

EditView (MAC OS 9): To access your sequence files from the CD or the file server (to be implemented later this spring), you will need the software program Edit View 1.0.1 and a conversion utility. Both can be downloaded at no cost from the Applied Biosystems web site.

Go to http://www.appliedbiosystems.com/support/software/dnaseq/installs.cfm and look in the gray box on the right. Click on Edit View 1.0.1 to download the program.

To download the conversion program go to http://www.appliedbiosystems.com/support/software/3100/conversion.cfm. Next to the program name you will see the word "MAC" in a blue box. Click on this box to download the conversion program.

Save your sequences from the file server into a new folder "My Sequences" on your hard drive.

Occasionally, the sequence files will be locked and need to be unlocked before they can be converted. (We don't know why or how they become locked, but they can.) To overcome this problem, open the "My Sequences" folder on your hard drive and highlight any one of the sequence files. On the menu bar go to "file", then to "get info" and select "general information." Look in the box on the bottom left corner of the window and make sure the "locked" box is not checked. If the file is locked, click on the "locked" box to uncheck it and unlock the file. If one sequence is locked, then it is likely that all sequence files will locked will have to be unlocked in this manner before conversion.

To start the conversion program, double click the conversion program icon. Double click on the "sample file win to mac" icon. This opens a window in the conversion program asking you where the files to convert are located. Highlight (do not open) the new folder "My Sequences" on your hard drive. Click the "choose" button. This will simultaneously convert all the files in the folder to Mac format.

Double click on the individual sequence file you wish to open and Edit View will open it automatically.

Edit View opens the electropherogram for the file first. In the bottom left corner of this window there are four buttons. The second button from the left is labeled GATC. Clicking this button will switch to a window showing the sequence data in nucleotide format. To toggle back to the electropherogram, click on the far right button. You should be able to print either window. You may also use select all and copy options to paste your nucleotide sequence into other programs. Subsequent sequence data files may be opened by selecting "file" and "open" on the menu bar.

4Peaks (MAC OS X):
Go to here to download the free version for molecular biologist to visualize and edit their DNA sequence files.

If you have any questions about using the programs, feel free to contact the Core Facility.

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Useful Links :

www.appliedbiosystems.com

www.usbweb.com Exo SAP-IT kit

www.qiagen.com

www.edgebio.com/

http://www.technelysium.com.au/chromas.html Chromas program -PC
( Freeware version 1.45 or V2.21 ( $39.00 ), See section above for instructions on how to use this program to view your results.

http://www.appliedbiosystems.com/support/software/dnaseq/installs.cfm Edit view 1.0.1 electropherogram viewer for the MAC. See instructions above on how to download Edit View and the conversion program to view your results.

ABI Documents on Demand . Enter the following part number in the box 'Part/Stock/Document Number' in order to receive the corresponding document:

1. Big Dye Terminator Cycle V3.1 Sequencing Kit Protocol, Rev A: 4337035A

2. Automated DNA Sequencing Chemistry Guide part number: 4305080

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