CEQ 2000 Experiments

Concluded Studies:

  • Selection of Quality Threshold Value (RL, error)

  • Tuning of pUC118 vs. General Samples

  • Sample handling dependent variations

  • Effect of Salt

  • Effect of DNA concentrations

  • Effect of Ethanol

  • Effect of Histidine, Cysein, Glycine

  • Read Length vs. start time

  • Sequencing of difficult regions

  • cDNA (heart) sequencing

  • Universal primers

  • Variations in formamide

  • ABI vs. CEQ 2000

  • Optimization of PCR protocols

Major Conclusions:

-We have found that the optimum quality value to export data is 0.9-0.95.

-The limitations set by the amount of salt, DNA, and ethanol in the cycle sequencing reaction or the ready load sample has been set by titration experiments. Sample sequencing results are considerably better when there is no salt or ethanol in the sample.

-Sequencing success does not necessarily depend on the DNA concentration in the cycle sequencing reaction. Total sample failure is due to effects that are not a function of concentration, ie, loss of pellet, salt, etc.

-M13 forward primer works much better in our samples than M13 reverse.

-Read length is a strong function of the start value.

-The CEQ 2000 was able to successfully sequence cDNA library.

-To facilitate efficient and large-scale genotype analysis, we are developing a genotype method using fluorescence-labeled universal primer and the CEQ 2000 system. Using the CEQ 2000 system elimintates the use of hazardous radioisotopes, autoradiograph or silver staining fluorescence labeling of primers and allows for multiplex PCR and STR profiling.

-Buffers made of histidine, cysein, and glycine are often used to increase the mobility of DNA. We attempted to use these buffers in our sequencing samples to measure their effect on the sample injected. No real effect was found.

Future and Current Studies:

  • Currently sequencing and assembling Bac clone of ~100kb in size.
  • Sequence human Pac clone (from human chromosome 15) of ~100kb in size.
  • Demonstrate SNP discovery using CEQ 2000.
  • Complete protocol optimization for simple prep methods.
  • Work on reduction for total failures.
  • Develop more recommendations for user friendliness.
  • Investigate all variables using 'real' samples, to maximize window of success for 'real' samples.