Showing papers by "Michael McClelland published in 1991"

Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping.

Abstract: Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between strains of almost any organism. We applied the technique to the mouse (Mus musculus). The characteristic differences in the AP-PCR genomic fingerprints between strains will be of value in strain identification and verification. Using one primer, we genetically mapped four polymorphisms in a set of C57BL/6J x DBA/2J recombinant inbreds. One of these polymorphisms is a length variant. The method will allow rapid genetic mapping of DNA polymorphisms without Southern blotting.

Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers.

Abstract: Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between slightly divergent strains of any organism Single oligodeoxyribonucleotide (oligo) primers have been used to generate such fingerprints, with the same primer being present at the 5' end of both strands for every PCR product We used three arbitrary oligos, individually and in pairs, to generate six different genomic fingerprints of the same mouse genomic DNAs Fewer than half of the products in genomic fingerprints generated using the oligos in pairs were the same as those produced by AP-PCR using one of the three oligos alone Thus, a few oligos could be used in a very large number of single and pairwise combinations, each producing a distinct AP-PCR fingerprint with the potential to identify new polymorphisms For example, 50 oligos can be used in a matrix of pairwise combinations to produce 2,500 fingerprints, in which at least half the data can be expected to be unique to each pair We demonstrate this principle by using two oligos, alone and together, to generate three sets of fingerprints and map thirteen polymorphisms in the C57BL/6J x DBA/2J set of recombinant inbred mice

Genomic fingerprints produced by PCR with consensus tRNA gene primers

Abstract: The polymerase chain reaction using only a single 'consensus' tRNA gene primer, or a pair of primers facing outward from tRNA genes, amplifies a set of DNA fragments in bacterial, plant and animal genomic DNAs. Presumably, these PCR fingerprints are mainly derived from the regions between closely linked tRNA genes. The pattern of the PCR products is determined by which genomes and which primer(s) are used. Genomic fingerprints are largely conserved within a species and, in bacteria, most products in the fingerprint are conserved between closely related species. Thus, PCR with tRNA gene consensus primers helps to identify species and genera.

Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction (AP-PCR).

Abstract: Using a novel procedure based on the polymerase chain reaction, we have developed a rapid, efficient, and economical method for identifying plant genotypes. The arbitrarily primed polymerase chain reaction (AP-PCR) generates reproducible fingerprints from any organism, without the need for DNA sequence information. These fingerprints include DNA fragment polymorphisms that can be (1) used for varietal identification and parentage determination, (2) followed in segregating populations produced by crosses, (3) used as markers for the construction of genetic maps, and (4) used to generate dendograms of phylogenetic relationships, especially at the intraspecific level. AP-PCR requires only minute quantities of DNA (10–25 ng per reaction) and therefore can be used in situations in which DNA is limiting. We demonstrate the use of AP-PCR to identify inbred parents of hybrid maize plants in double-blind experiments.

Site-specific methylation: effect on DNA modification methyltransferases and restriction endonucleases

Abstract: We present in Table I an updated list of the sensitivities of over 240 restriction endonucleases to the site-specific DNA modifications '14C, n-6C, h1"5C, and m6A, four modifications that are common in DNA prokaryotes, eukaryotes, and their viruses (Mc2,Mc5,Mc8,Mcl 1 ,Ne3,Ne4). Table II is a list of over 130 characterized DNA methyltransferases. A detailed list of cloned restrictionmodification genes has been made Wilson (Wi4). Table m lists the sensitivities of over 20 Type II DNA methyltransferases to m4C, nSC, hm5C, and m6A modification. Most DNA methyltransferases are sensitive to non-canonical modifications within their recognition sequences (Bu5,MclO,Ne3,Po4), and this sensitivity may differ from that of their restriction endonuclease partners. Finally, several restriction endonuclease isoschizomers are known to differ in their ability to cleave DNA which has been methylated. Table IV lists over 20 known isoschizomer pairs and one isomethylator pair, along with the modified recognition sites at which they differ.

Electrophoretic separation of the three Rhizobium meliloti replicons.

Abstract: The megaplasmids and the chromosome from the bacterium Rhizobium meliloti 1021 were separated in preparative quantities by using transverse alternating-field gel electrophoresis. The genetic content of each electrophoretically separated band was determined by Southern hybridization with replicon-specific probes and by comparison with Agrobacterium tumefaciens transconjugants harboring either pSym-a or pSym-b megaplasmids. Pulsed-field gel electrophoresis analyses of PacI (5'-TTAATTAA-3') and SwaI (5'-ATTTAAAT-3') digests of the whole genome and of the separated replicons were used to calculate genome sizes in two R. meliloti strains. In these strains, PacI digestion yielded only four fragments for the entire genome. The sizes of the PacI fragments from R. meliloti 1021 in megabase pairs (Mb) were 3.32 +/- 0.30, 1.42 +/- 0.13, 1.21 +/- 0.10, and 0.55 +/- 0.08, for a total genome size of 6.50 +/- 0.61 Mb. Southern hybridization with replicon-specific probes assigned one PacI fragment to the chromosome of R. meliloti 1021, one to pRme1021a, and two to pRme1021b. PacI digestion of A. tumefaciens pTi-cured, pSym transconjugants confirmed these assignments. In agreement with PacI data, the addition of the six SwaI fragments from R. meliloti 1021 gave a genome size of 6.54 +/- 0.43 Mb. pRme1021a was calculated to be 1.42 +/- 0.13 Mb, 1.34 +/- 0.09 Mb, and 1.38 +/- 0.12 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021a, respectively. pRme1021b was calculated to be 1.76 +/- 0.18 Mb, 1.65 +/- 0.10 Mb, and 1.74 +/- 0.13 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021B, respectively. The R. meliloti 1021 chromosome was calculated to be 3.32 +/- 0.30 Mb, 3.55 +/- 0.24 Mb, and 3.26 +/- 0.46 Mb on the basis of PacI data, SwaI data, and the migration of uncut chromosome, respectively.

PCR with 5-methyl-dCTP replacing dCTP

Abstract: When dCTP is replaced by methyl5-dCTP in the polymerase chain reaction some templates cannot be efficiently amplified by Taq polymerase or Vent polymerase using standard cycling parameters. However, this phenomenon can be overcome by increasing the temperature of the denaturation steps to 100 degrees C, or by adding dITP to destabilize the m5dC:dG base pairs. Once the block to amplification of m5dC-substituted DNA was overcome, methylated DNA from the 'superpolylinker' of the plasmid pSL 1180 was used as a substrate to check the methyl-sensitivity of a variety of restriction endonucleases. The m5dC-substituted DNAs should also be valuable substrates for defining the specificity of methyl-dependent endonucleases.

Restriction fragment fingerprint and genome sizes of Staphylococcus species using pulsed-field gel electrophoresis and infrequent cleaving enzymes.

Abstract: Large restriction fragments of genomic DNA from Staphylococcus species were separated by pulsed-field gel electrophoresis (PFGE). Five different strains of S. aureus (ISP8, SAU3A, PS96, ATCC 6538, ATCC 15564) and three representative strains of S. haemolyticus SM102, S. warneri MCS4, S. cohnii LK478 from human hosts, and one strain of S. aureus (ATCC 8432) from an avian host were used in this study. Since Staphylococcus is A + T rich (approximately 67%), restriction fragments were obtained by digesting chromosomal DNA with endonucleases that recognize GC-rich sequences. Five enzymes Csp I, Sma I, Ecl XI, Ksp I, or Sac II were used for generation of few (7 to 16) distinctly separated fragments, with average sizes in the range of 200-300 kb. The size distribution of restriction fragments for each enzyme for each strain produced a strain-identifying fingerprint, and the genome size of each strain was determined from such restriction fragments separated by PFGE.

Enzymatic cleavage of a bacterial chromosome at a transposon-inserted rare site

Abstract: The sequential use of the methylase M.Xbal (5'.TCTAGm6A) and the methylation-dependent endonuclease Dpnl (5'-Gm6A decreases TC) results in cleavage at 5'.TCTAGA decreases TCTAGA. This recognition sequence was introduced into a transposon derived from the Mu bacteriophage and transposed into the genome of the bacterium Salmonella typhimurium. M.Xbal methylation was provided in vivo by a plasmid containing the M.Xbal gene and the S. typhimurium genome was cleaved to completion by Dpnl at one or more sites, depending on the number of transposon insertions. The resulting genomic fragments were resolved by pulsed-field electrophoresis. The potential use of single M.Xbal/Dpnl cleavage sites as reference positions to map rare restriction sites is discussed.