Analogs of guanine nucleoside triphosphates for sequencing applications.

We have synthesized more than 30 different deoxyribonucleosides and triphosphates with modifications either in the base or the phosphate moiety as analogs of 2'-dGTP for DNA sequencing applications. All the modified nucleoside triphosphates were tested as substrates for DNA polymerases, including Sequenase T7 DNA polymerase or Thermo Sequenase DNA polymerase. Two of the analogs, 7-ethyl-7-deaza-dGTP and 7-hydroxymethyl-7-deaza-dGTP meet our requirements as better sequencing reagents.

Uniform band intensities in fluorescent dye terminator sequencing.

The use of Cyanine dye (Cy5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.

Thermo Sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing.

A combination of thermostable enzymes has been developed that produces higher quality cycle sequences. Thermo Sequenase DNA polymerase is a thermostable enzyme engineered to catalyze the incorporation of ddNTPs with an efficiency several thousandfold better than other thermostable DNA polymerases. Since the enzyme also catalyzes pyrophosphorolysis at dideoxy termini, a thermostable inorganic pyrophosphatase is needed to remove the pyrophosphate produced during sequencing reactions. Thermoplasma acidophilum inorganic pyrophosphatase (TAP) is thermostable and effective for converting pyrophosphate to orthophosphate. The use of the combination of Thermo Sequenase polymerase and TAP for cycle sequencing yields sequence data with uniform band intensities, allowing the determination of longer, more accurate sequence reads. Uniform band intensities also facilitate interpretation of sequence anomalies and the presence of mixed templates. Sequencing PCR products of DNA amplified from heterozygous diploid individuals results in signals of equal intensity from each allele.

Enzyme analysis of mouse extra-embryonic tissues.

We have separated for enzyme analysis the following layers that surround the conceptus at midgestation: decidua, trophoblast, parietal endoderm (including Reichert's membrane), visceral endoderm, yolk-sac mesoderm and amnion. Measurement of several catabolic enzyme activities (N-acetyl-beta, D-hexosaminidase, beta-glucuronidase, alkaline and acid phosphatases and non-specific esterases) in these tissues indicates that they are biochemically distinct, perhaps reflecting the different functions that they perform in providing the embryo proper with a desirable environment for differentiation and development. Our studies also provide an example of how visceral endoderm cells can effectively block passage of maternal macromolecules (in this case a serum esterase) in the fetal circulation. Finally, since there is often difficulty in distinguishing among early embryonic and extra-embryonic cell types produced in teratocarcinoma cultures, we have considered how our observations might be of use in the respect, particularly in discriminating between visceral and parietal endoderm.

In vitro development of core cells of the inner cell mass of the mouse blastocyst: effects of conditioned medium.

Blastocysts submitted to two rounds of immunosurgery give rise to cores of presumptive ectoderm cells, many of which do not survive for more than 48 hours when cultured individually. Precoating of the culture plates with conditioned medium (CM) from PYS-2 cells increases the incidence with which cores regenerate an outer layer. This procedure also improves the survival frequency of the cores, but only for a limited period of time. The small number of cores which survive for two weeks or more, either in uncoated or CM-coated plates, give rise to any array of cell types, including giant cells resembling trophoblast.