AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns.

Taq DNA polymerases in which the phenylalanine is substituted by a tyrosine at position 667 (Taq F667Y) are members of a new class of DNA polymerases that incorporate chain-terminating dideoxyribonucleoside triphosphates (ddNTPs) much more efficiently than the wild-type Taq DNA polymerase. Improved incorporation of ddNTPs into DNA during cycle sequencing using AmpliTaq DNA polymerase, FS (Taq-FS, a member of the Taq F667Y family), and dye-labeled primers results in nearly uniform peak heights in the sequencing trace. This is not the case when dye-labeled ddNTPs are used in Taq-FS cycle sequencing reactions. While the rate of dye-terminator incorporation is more efficient with Taq-FS, the peak pattern is still highly variable and different from that produced by the wild-type enzyme. We have systematically examined pairs of sequence-tagged sites that vary at only a single nucleotide to determine how base changes influence the peak heights of neighboring bases in sequencing traces generated by the Taq-FS dye-terminator chemistry. In 31 of 64 possible 3-base windows (48%), we find that the peak height of a particular base can be predicted by knowing just one or two bases 5' to the base in question. We have also compared and contrasted the peak patterns produced by the Taq-FS enzyme with those previously identified for the wild-type enzyme. Establishing the patterns in peak heights within local sequence contexts can improve the accuracy of base-calling and the identification of polymorphisms/mutations when using the Taq-FS dye-terminator cycle-sequencing chemistry.

Peak height variations in automated sequencing of PCR products using Taq dye-terminator chemistry.

Direct sequencing of PCR products using Taq DNA polymerase with dye-labeled dideoxy chain terminators results in traces with uneven peaks. The peak height variations reflect the disproportionate rate of incorporation of deoxynucleotides vs. their analogs, a phenomenon that is highly dependent on the neighboring DNA sequence. Such peak height variations make it difficult to call bases correctly or to interpret whether or not a polymorphism is present. We have systematically examined pairs of sequence-tagged sites that vary at only one nucleotide to determine how a single base change will affect the peak heights of neighboring bases. We have found that the peak height of a particular base can often be predicted from the knowledge of just one or two nucleotides 5'- to the base in question. We have also observed several artifacts that occur consistently in the sequencing traces. These artifacts can be misinterpreted as polymorphisms or can obscure the real peak at that site. The empirically derived trends presented in this report can be utilized profitably when one is editing sequence data or examining them for polymorphisms and mutations.

Fluctuating asymmetry of morphometric characters in house mice: the effects of age, sex, and phenotypical extremeness in a randombred population.

Fluctuating asymmetry (FA) was calculated for nine paired morphometric characters in a randombred population of house mice in order to assess the effects of age, sex, and phenotypical extremeness. FA did not significantly vary between sexes for any of the characters, and age proved to be significant only for innominate length. Additionally, FA values in high extreme individuals and low extreme individuals were compared to those for intermediate individuals to discover whether extreme individuals showed increased fluctuating asymmetry (decreased developmental stability) due to increased levels of homozygosity. Differences in fluctuating asymmetry magnitudes were found between the groups, although the extreme individuals did not consistently show increased fluctuating asymmetry and FA differences also showed no significant association with the level of heritability of each character. Potential reasons for these findings are discussed.

Central body of the Azotobacter cyst.

Parker, Laura T. (Louisiana State University, Baton Rouge), and M. D. Socolofsky. Central body of the Azotobacter cyst. J. Bacteriol. 91:297-303. 1966.-Sodium citrate was found to effect extensive rupture of cyst coats of Azotobacter vinelandii. By filtering a citrate-ruptured cyst suspension through a Millipore microfiber glass prefilter, a preparation of viable central bodies was obtained that contained less than 1% residual cysts and vegetative cells. Electron micrographs showed the central bodies to have a cell wall and cell membrane. Free central bodies germinated into typical vegetative cells. Central bodies exhibited approximately the same resistance to ultraviolet radiation, sonic treatment, and elevated temperatures as did vegetative cells; cysts were much more resistant. Manometric experiments indicated that central bodies and cysts have almost the same oxidative capabilities. Results of resistance studies indicated that the central body is a contracted vegetative cell encased in a protective coat. The cyst coat appears to account for the resistance of the cyst.