Factors Which Affect Sequencing Quality
The primary factors which affect the quality of a sequencing runs are: 1) DNA quality and amount; 2) Primer quality and amount; 3) Ratio of primer to template; 4) Effective hybridization of primer to DNA; and 5) Actual sequence of DNA template. If poor sequencing data results are obtained, the facility staff is ready to help troubleshoot the problem and/or put you in contact with other University investigators who consistently have had success with this technology.

Quality Control
Each gel run includes two forms of standards or internal controls. A lane of "marker" DNA fragments is run to determine if the instrument is efficiently picking up the dye signals and if the individual lanes are correctly tracked. Several lanes containing actual sequencing reactions (performed simultaneously with the batch of investigator samples) on M13 (ss) DNA and/or pGEM (ds) are run to determine the efficacy of the sequencing reagents and enzyme. Thus, if an investigator's sample does not produce satisfactory sequence results the problems can usually be localized to the primer or template. Often, the resultant electrophoretograms from sequencing runs can be used to diagnose the problem, i.e. insufficient DNA, poor quality DNA etc. and therefore close consultation with facility staff as to apparent problems of this kind is important to remedy the situation.

Primer Selection and Priming Artifacts
Desirable primer-template interaction is based on many parameters. The length, base composition, and sequence of the primer determine the conditions used to denature and anneal in a cycle sequencing procedure. In a core facility setting such as ours, a standard set of conditions is generally employed, which precludes tailoring to the needs of individual samples in terms of reaction conditions. Therefore users are advised to select or design their primers that meet some simple requirements. In general, primers should be selected to have one or more G- or C-bases at the 3'-end, a base composition of approximately 55% GC- content and no inverted repeats or homopolymeric regions. The manufacturer provides some guidelines in primer selection which are listed in Appendix C. A computer program for primer design (Oligo 5.0) is available in the facility. This program has proven useful for primer design in several University laboratories

When a DNA sequencing strategy, such as primer walking is involved, priming site selection is important in order to achieve a balance of appropriate redundancy and assured quality of the resultant sequencing data. Priming sites should be selected only from unambiguous sequence regions. Mismatches between the primer and the primer binding site can reduce the stability of the complex formed, and therefore adversely affect the product extension.

With proper sample preparation and the correct template/primer ratio, the BigDye terminator method regularly yields 700-800 bases of sequence information with > 98% accuracy for single-stranded or double-stranded DNA.

It is also important to remember that the first 20-40 bases from the 3'-end of the primer may not yield reliable sequence data. Therefore, priming sites should be choosen to take that into account so that critical sequence information from an important region of the template will not be unreliable.

Some DNA's, due to their intrinsic nature, are unsuitable for sequencing with the DyeDeoxy termination chemistry. Users are reminded that several other chemistries might be more efficient for their particular applications. These include Dye primer cycle sequencing, Sequenase terminator single-stranded DNA sequencing, Sequenase terminator double-stranded DNA sequencing and Sequenase primer single stranded DNA sequencing, to name but a few. Users are encouraged to consult with us when problem sequences are likely to be incurred.

Data Disposition
All forms of data, from the crude, color electrophoretogram, to the edited sequence is available to the investigator based on his or her preference. Generally, hardcopy of the base-calling electrophoretigram and the resultant sequence is all the information typically needed by the investigator. An example of such data as well as a Sequencing Request Form, are seen in Appendix D. However, if the investigator desires, the crude data can be stored on a computer disk of the investigator's and given to him. All crude data is stored in the laboratory on an optical drive and will be kept for a period of 1 year. The sequence read-out can also be directly sent via email to investigators if desired.

Data Viewing and Manipulation
Programs are available in the facility for sequence assembly and handling and investigators are welcome to use the computer in the facility to work with their data. Depending on the demand, identical programs will be made available in the ITC for their use there.

General Comments
Investigators typically desire one of two types of information; complete sequence of a segment of DNA or the sequence of a particular, localized region, such as a mutagenesis site, or insertion site etc. Where maximum accuracy is required, we recommend that sequencing be performed on both DNA strands and that multiple, simultaneous sequencing reactions be performed

If an investigator wishes to initiate a large-scale sequencing effort, such as primer walking, we suggest they discuss this with the staff so we can better coordinate our efforts to meet their needs. An example of such an effort would be in primer design and synthesis by the facility.

Finally, as in all of our efforts in the facility, the closer the investigators work with the staff, the higher the likelihood of success and satisfaction with the facility. Therefore we encourage the investigators to come and meet with the facility staff and discuss their needs and how we can best serve them.

For additional information on this service please contact Dr. Yongde Bao, 982-2551.