The recA gene of Borrelia burgdorferi.

The nucleotide sequence of the Borrelia burgdorferi (Bb) Sh-2-82 recA gene has been determined using PCR-based approaches without the construction of a genomic library. The gene should encode a protein of 365 amino acids which is highly homologous to other known RecA proteins. It represents a new homolog from a distinct phylogenetic branch of eubacteria. Although, previous reports concluded that recA is absent from Bb, the identification presented here conclusively shows its presence and reaffirms the ubiquity of RecA in prokaryotes.

Reloading and stability of polyacrylamide slab gels for automated DNA sequencing.

Current automated fluorescent instruments are all based on slab gels that are used once and then discarded. Practitioners of capillary gel electrophoresis often reuse their gels for multiple samples. As slab gels are made thinner to increase speed, the ability to reload new samples after each run will become more desirable. Techniques have previously been developed for reloading and stabilizing capillary gels. The application of these methods to slab gel electrophoresis is reported. Gels are shown to be reusable for at least four consecutive automated runs. The stability of various slab gel formulations and their ability to survive multiple loadings with sequencing samples are compared. Formamide-containing gels are shown to be superior to their urea counterparts. The potential that running buffer additives have for improving automated DNA sequencing is discussed. Residual template DNA in sequencing samples can produce gel instability, reduce resolution and decrease signal. These effects are examined.

Rapid cycle sequencing in an air thermal cycler.

Cycle sequencing using Taq DNA polymerase has gained popularity recently due to reduced template requirements, improved signal and its ability to directly sequence PCR fragments. A major drawback to the technique is the time required for performing reactions in a block-based thermal cycler. To help cycle sequencing compete with other methods, we have modified the protocol to be performed in capillaries using an air-based thermal cycling instrument. This instrument has been developed and optimized for rapid, specific amplification of DNA by PCR. The resulting cycle sequencing methodology is faster than block-based approaches; a reaction can be completed in 25 min, compared with about 2 h in a conventional instrument. Thus, the speed of the technique is competitive with standard uncycled T7 or Taq reactions. Accuracy of the sequencing data is improved; two problem areas in the sequence obtained with a block cycler are ameliorated by the capillary methodology. This technique represents a novel approach to cycle sequencing that will further the development of capillary-based analytical methods.

Stability of capillary gels for automated sequencing of DNA.

Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Extensive mitochondrial diversity within a single Amerindian tribe.

Sequencing of a 360-nucleotide segment of the mitochondrial control region for 63 individuals from an Amerindian tribe, the Nuu-Chah-Nulth of the Pacific Northwest, revealed the existence of 28 lineages defined by 26 variable positions. This represents a substantial level of mitochondrial diversity for a small local population. Furthermore, the sequence diversity among these Nuu-Chah-Nulth lineages is greater than 60% of the mitochondrial sequence diversity observed in major ethnic groups such as Japanese or sub-Saharan Africans. It was also observed that the majority of the mitochondrial lineages of the Nuu-Chah-Nulth fell into phylogenetic clusters. The magnitude of the sequence difference between the lineage clusters suggests that their origin predates the entry of humans into the Americas. Since a single Amerindian tribe can contain such extensive molecular diversity, it is unnecessary to presume that substantial genetic bottlenecks occurred during the formation of contemporary ethnic groups. In particular, these data do not support the concept of a dramatic founder effect during the peopling of the Americas.