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  • Why is my sequencing not working?
  • Why am I getting 300 bp read length rather than 600 bp read length?
  • Take a look of the TroubleShooting Page

Overview of DNA Sequencing

Template Preparation

Primers Available

Interpret Your Sequencing Data


1. Overview of DNA Sequencing

The sequencing method used in our facility is automated fluorescent dye-terminator cycle sequencing, based on the chain-termination dideoxynucleotide method of Sanger and coworkers. The automated sequencing method, as developed by ABI Perkin-Elmer for use on the ABI 377 Sequencer, uses PCR to incorporate dideoxynucleotides which contain fluorescent dyes in a primer extension sequencing reaction.

The PCR reaction consists of DNA template, primer, a special DNA polymerase, unlabeled dNTP's, fluorescently labeled ddNTP's, and appropriate buffer in ultrapure sterile water. All customer samples are run using the same standard PCR cycle on a GeneAmp 9600. During the PCR reaction, the double stranded DNA template is denatured one time, before the cycles start, for 2 min. at 96oC. After this "hot start", the temperature cycle begins with denaturation at 96oC for 10 sec., a ramp down to 50oC for 15 sec. to allow the primers to anneal to the template's priming site, then a ramp up to 60oC for 4 min. to allow extension of the primer by the polymerase. This temperature cycle is repeated for 25 cycles. The DNA polymerase used is "Amplitaq FS", a thermostable modified form of Thermus aquaticus DNA polymerase which is produced in E. coli. Amplitaq has no 3'-5' exonuclease activity, enabling rapid and efficient incorporation of dNTP's and the fluorescent ddNTP's.

When the PCR is complete, the reaction mix contains a population of PCR fragments of different lengths, each terminating in a fluorescent-dye-containing dideoxynucleotide. Each dideoxynucleotide base contains a different fluorescent dye which emits a characteristic wavelength, thus the identity of the dye corresponds to the final base on that fragment. The entire reaction is purified, then run in a single lane on a polyacrylamide gel in an ABI 377 Sequencer, so that the fragments separate according to size. As the fragments are electrophoresed, they run past a laser detector at the bottom of the gel, and the emission wavelength of each fragment is detected.

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The data is compiled into a gel image, analyzed by ABI software and the resulting sequence is written into a text file and a chromatogram file.

 



2. Template Preparation

DNA must be very pure for automated sequencing. Residual salts, EtOH, EDTA or organics in the tube will inhibit the sequencing reaction and result in poor data. See Suggested Preparation Methods

3. Primers Available

Primer Name Sequence
Universal (M13 forward) 5' TG-TAA-AAC-GAC-GGC-CAG-T 3'
M13 reverse 5' CA-GGA-AAC-AGC-TAT-GAC-C 3'
SP6 5' GAT-TTA-GGT-GAC-ACT-ATA-G 3'
T7* 5' AAT-ACG-ACT-CAC-TAT-AGG 3'
T7 promoter 5' TAA-TAC-GAC-TCA-CTA-TAG 3'
T7 terminator 5' CTA-GTT-ATT-GCT-CAG-CGG 3'
T3 5' ATT-AAC-CCT-CAC-TAA-AGG 3'
KS 5' TCG-AGG-TCG-ACG-GTA-TC 3'
SK 5' CGC-TCT-AGA-ACT-AGT-GGA-TC 3'
*: Please note that some vectors have T7 sites which are not completely complementary with this standard primer. Please double check your vector sequence when using this primer.
Primer Design Considerations
Good sequencing primers are generally 18-20 bases long, have around 50% GC content and Tm's between 55 and 75 degrees. Use of an oligo design computer program can greatly simplify designing primers. CSUPERB and the Microchemical Core Facility have purchased an oligonucleotide design program OLIGOTM for the use of SDSU faculty, students and staff. This program is installed on a Mac in the Core and on the Fluorimager Mac in LS334. The manual can be checked out in LS301.

4. Interpret Your Sequencing Data
  • Our lab routinely produces high quality data with read lengths to 600 bases; however, this depends on many factors, including how clean the template DNA is, or the annealing efficiency of the primer. To prepare DNA which is clean enough for automated sequencing, see Template Preparation.
  • Other problems in the sequencing reaction can affect success as well. Some examples of these, with their solutions, follow.



MCF Home SDSU CSUPERB Contact Us Last Updated: 5/2006