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M A Lodhi

Publications and source records attributed to M A Lodhi.

4 recordsLinked to original sources
PubMed1996

Fluorescence-based sequencing of double-stranded DNA by hexamer string priming.

A fluorescence-based, T7 (Sequenase) dye terminator method for sequencing double-stranded DNA using strings of three contiguous hexamers as primers and single-stranded binding protein is described. In this method, the circular, supercoiled DNA vector pUC19 is first linearized with a restriction enzyme to create a sequenceable template. Sequencing is then accomplished using three cycles of "denaturation," annealing, and extension/termination. Twenty-two of 33 hexamer strings tested in a controlled study produced acceptable sequence, with read lengths varying from the mid 300s to the low 400s and a base-calling accuracy of at least 97%. To test its potential utility in directed DNA sequencing, the protocol was then used to completely sequence both strands of pUC19. For this test project, a total of 28 hexamer strings was used with an overall successful priming rate of 75%. The current protocol appears to be sufficiently robust to be used in the finishing phase of a shotgun sequencing project and is amenable to semiautomation. Prospects for using the protocol for full-scale directed sequencing as well as for full automation are discussed.

Automation↗
PubMed1996

High-quality automated DNA sequencing primed with hexamer strings.

The finishing phase of genome sequencing projects is expensive, in part, because of the cost of de novo synthesis of custom primers and the management burden associated with obtaining and using them for primer walking. One approach to reduce these high costs is the use of a presynthesized library of short oligonucleotides (8-10 bases) rather than long primers. The use of such a library eliminates the need for custom synthesis of oligonucleotides, providing the convenience of priming from any site by combining two to three short oligonucleotides to form a string with the required specificity. The first practical implementation of this strategy presented a robust protocol for using hexamer strings with radioisotopic labelling. Whereas versions of this technique have subsequently been implemented on fluorescent sequencers we felt that there was a need to develop and extensively test a protocol that consistently gave read lengths comparable to dye-terminator sequencing with longer primers. We have developed a new two-cycle fluorescent Sequenase terminator procedure for using hexamer strings. We tested this procedure using a set of 32 different 3 hexamer primer strings, each known to be functional to some degree in radioisotopic sequencing, on single-stranded M13mp18 template and ABI 373 DNA sequencers. The overall success rate of priming with these hexamer primer strings is 97% with the failure of only one string. In this case, the corresponding 18-mer primer also failed to produce usable sequence from M13mp18 template. The average read length from reactions successfully primed with the 31 different hexamer strings was 461 bases with > 99% base-calling accuracy. The current protocol is robust enough to be used in virtually any situation where primer walking on single-stranded templates is used. The success rate and read lengths make it universally applicable to the sequencing of single-stranded templates on automated sequencers. It is also amenable to automation.

Automation↗
PubMed1995

A molecular marker based linkage map of Vitis.

Genetic linkage maps of Vitis (2n = 38) have been constructed from a single interspecific hybrid grape population (60 seedlings) of 'Cayuga White' X 'Aurore'. The maps were primarily based on 422 RAPD markers but also included 16 RFLP and isozyme markers. These maps had an average distance of 6.1 cM between markers and were developed using a double-pseudotestcross strategy. The 'Cayuga White' map had 214 markers covering 1196 cM and that of 'Aurore' spanned over 1477 cM with 225 markers. The 'Cayuga White' map consisted of 20 linkage groups, whereas 22 linkage groups comprised the 'Aurore' map. The number of groups reduced to 19, as in some instances two or more groups from one parent showed homology with a single group from the other parent on the basis of markers heterozygous in both parents. Each linkage group ranged in size from 14 to 135 cM in 'Aurore' and from 14 to 124 cM in 'Cayuga White'. These maps provide enough coverage of the genome to allow quantitative trait locus analysis and map-based gene cloning.

Base Sequence↗