Waikato DNA Sequencing Facility (WDSF)
The types of DNA template which can be sequenced are double and single-stranded plasmids, PCR products, cosmids and artificial chromosomes. Regardless of template type, it is important to have a good quality template that is homogeneous and free of contaminating salts, solvents, RNA, proteins and chelating agents.
With quality templates we routinely achieve read lengths of greater than 700 bp. Good sequencing results are obtained from template purified using commercially available kits or carefully executed alkaline lysis and PEG precipitation protocols. We have available the recommended procedures for preparation of templates. If you should require these protocols, please do not hesitate to contact us.
Concentrations measured using standard spectrophometric techniques will only be accurate for highly pure DNA. Please only use a spectrophotometer reading where the A260/A280 ratio is >1.8. Impurities such as proteins, oligonucleotides, and unincorporated nucleotides prevent accurate spectrophometric measurements and the actual yield can be significantly lower than the concentration indicated.
For double-stranded DNA, ethidium bromide fluorescence on an agarose gel may be inaccurate as its fluorescence is not specific to double-stranded DNA. A mass ladder should be used for comparison in this method.
We quantitate DNA using H-33258 fluorescence enhancement, which is highly sensitive, specific and accurate. Whilst this method provides accurate quantitation of DNA even in the presence of RNA, protein, nucleotide, dilute detergents and protein denaturants, the presence of any of these factors will adversely affect the sequencing reaction.
The WDSF takes all due care when undertaking the testing of DNA templates but does not accept liability for any failed reactions that occur due to:
»Incorrect DNA concentrations given;
»Incorrect Primer concentration supplied;
»Failure to provide necessary or accurate information;
»Poor Template or Primer quality.
Control reactions are regularly run and if the WDSF is in any doubt as to the cause of failed or low quality reactions, the reactions can be repeated provided sufficient primer and template is available.
Repeats and reruns are performed at the sole discretion of the WDSF
Causes of poor results may include, but are not limited to;
»Degraded or Heterogeneous material
»Contaminating Salts (such as Mg2+)
»Residual process buffers
»Chelating agents (especially EDTA)
Please remember that the Waikato DNA sequencing facility provides sequencing and genotyping for research and academic purposes only, and results should not be used for forensic, medical or diagnostic purposes.
The following is taken from pages 3-12 and 3-13 of the DNA Sequencing: Chemistry Guide Part Number 903563, Version A, May 1995. © Copyright 1995, The Perkin-Elmer Corporation.
Although base calling is easiest for the analysis software when signal strength is high, good signal strength does not always go hand-in-hand with high quality data. Background noise can obscure the true sequence data.
Dirty DNA templates are the most common cause of noisy data. Some template preparation procedures are not compatible with fluorescent sequencing. A template that gives poor sequence is usually said to be dirty if it contains some impurity that inhibits the sequencing enzyme or that inactivates some other reagent in the sequencing reaction. The contaminants are usually small molecular weight species, proteins, or RNA. Templates that produce poor data can sometimes be purified to a quality that yields good data. Please contact us if you require information on template clean-up techniques.
If two or more different DNA templates are present in the reaction (a mixed template) and all possess a primer annealing site, sequence data from each will be superimposed in the electropherogram giving a superimposed peak pattern.
It is a good idea to check each template preparation by agarose gel electrophoresis to determine its purity. When purifying recombinant plasmids in bacteria, plate out the transformants to obtain isolated colonies. Then select a single colony and restreak out on a plate to again select the colony for growth of cultures. If isolating bacteriophage DNA by growth from plaques, pick from a fresh plate and choose an isolate that is well away from others. If sequencing a PCR fragment, check the product on a gel to ensure that only one product was formed in the PCR or use a sequencing primer nested within those used for the amplification.
Enzyme slippage can occur in homopolymer regions (such as the poly(A) tails present in cDNA), thus skipping a base or incorporating an additional one. The sequence beyond the homopolymer region may then be shifted by one or more bases giving the appearance of multiple overlapping sequences in the electropherogram or stuttering peaks.
Lastly, multiple priming events (either by n-1 primers, by a different contaminating primer or a single primer annealing to more than one site) can lead to multiple overlapping sequences.
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