The Use of Short Adapters for Priming PCR of Unknown Chromosomal DNA Fragments

PCR Topics ◽  
1991 ◽  
pp. 53-55
Author(s):  
J. Schneider ◽  
H. Schrempf
Keyword(s):  
Dna Fragments ◽  
Chromosomal Dna

Effects of mutation position on frequency of marker rescue by homologous recombination

1992 ◽  
Vol 12 (8) ◽  
pp. 3609-3613
Author(s):  
L Jiang ◽  
A Connor ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Normal Function ◽  
Constant Region ◽  
Dna Fragments ◽  
Wild Type ◽  
End Effect ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Marker Rescue ◽  
Immunoglobulin Mu

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

Development of a fungal transformation system based on selection of sequences with promoter activity

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

scholarly journals Development of a fungal transformation system based on selection of sequences with promoter activity.

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305 ◽  
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

Horizontal Acquisition of Divergent Chromosomal DNA in Bacteria: Effects of Mutator Phenotypes

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 13-21
Author(s):  
Jeffrey P Townsend ◽  
Kaare M Nielsen ◽  
Daniel S Fisher ◽  
Daniel L Hartl
Keyword(s):  
Amino Acid ◽  
Genetic Variability ◽  
Environmental Dna ◽  
Fold Increase ◽  
Dna Fragments ◽  
Chromosomal Dna ◽  
Bacterial Populations ◽  
E Coli ◽  
Beneficial Effects ◽  
Acid Protein

Abstract We examine the potential beneficial effects of the expanded access to environmental DNA offered by mutators on the adaptive potential of bacterial populations. Using parameters from published studies of recombination in E. coli, we find that the presence of mutators has the potential to greatly enhance bacterial population adaptation when compared to populations without mutators. In one specific example, for which three specific amino acid substitutions are required for adaptation to occur in a 300-amino-acid protein, we found a 3500-fold increase in the rate of adaptation. The probability of a beneficial acquisition decreased if more amino acid changes, or integration of longer DNA fragments, were required for adaptation. The model also predicts that mutators are more likely than nonmutator phenotypes to acquire genetic variability from a more diverged set of donor bacteria. Bacterial populations harboring mutators in a sequence heterogeneous environment are predicted to acquire most of their DNA conferring adaptation in the range of 13–30% divergence, whereas nonmutator phenotypes become adapted after recombining with more homogeneous sequences of 7–21% divergence. We conclude that mutators can accelerate bacterial adaptation when desired genetic variability is present within DNA fragments of up to ∼30% divergence.

The use of male-specific chromosomal DNA fragments to determine the sex of bovine preimplantation embryos

1989 ◽  
Vol 31 (1) ◽  
pp. 95-104 ◽  
Author(s):  
K.R. Bondioli ◽  
S.B. Ellis ◽  
J.H. Pryor ◽  
M.W. Williams ◽  
M.M. Harpold
Keyword(s):  
Preimplantation Embryos ◽  
Dna Fragments ◽  
Chromosomal Dna ◽  
Male Specific

Chromosomol DNA fragments from mouse cells exposed to an intercalating agent contain a 175-kdalton terminal polypeptide

1985 ◽  
Vol 63 (7) ◽  
pp. 780-783 ◽  
Author(s):  
Ray K. Ralph ◽  
Ronald Hancock
Keyword(s):  
Electron Microscopy ◽  
Molecular Mass ◽  
Topoisomerase Ii ◽  
Relative Molecular Mass ◽  
Dna Fragments ◽  
Torsional Stress ◽  
Chromosomal Dna ◽  
Terminal Protein ◽  
Mouse Cells ◽  
Polypeptide Chains

A 175 kdalton (kDa) polypeptide is bound covalently to the chromosomal DNA fragments from mouse cells exposed to the intercalating agent 4′-[(9-acridinyl)-amino]methansulphon-m-anisidide. Electron microscopy shows a terminal protein on the DNA fragments, whose 5′-termini are blocked. Since the relative molecular mass of topoisomerase II polypeptide chains is also about 175 kDa and topoisomerase II inhibitors prevent intercalator-induced DNA fragmentation, we propose that the polypeptide bound covalently to the 5′-terminus of the DNA fragments is a polypeptide derived from frequently integrated topoisomerase II operating to normalize torsional stress resulting from intercalation.

scholarly journals Ultraaccurate genome sequencing and haplotyping of single human cells

2017 ◽  
Vol 114 (47) ◽  
pp. 12512-12517 ◽  
Author(s):  
Wai Keung Chu ◽  
Peter Edge ◽  
Ho Suk Lee ◽  
Vikas Bansal ◽  
Vineet Bafna ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Sequencing ◽  
Single Cells ◽  
Error Rates ◽  
Human Cells ◽  
Sequence Information ◽  
Dna Fragments ◽  
Chromosomal Dna ◽  
Haplotype Information

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10−8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs.

scholarly journals Restriction Endonuclease Analysis Discriminates Bordetella bronchiseptica Isolates

2000 ◽  
Vol 38 (12) ◽  
pp. 4387-4393 ◽  
Author(s):  
Randy E. Sacco ◽  
Karen B. Register ◽  
Gwen E. Nordholm
Keyword(s):  
Restriction Enzyme ◽  
Host Species ◽  
Restriction Endonuclease Analysis ◽  
Bordetella Bronchiseptica ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Dna Fragments ◽  
Chromosomal Dna ◽  
Agarose Gels ◽  
Endonuclease Analysis

One hundred ninety-five Bordetella bronchisepticaisolates from 12 different host species worldwide were characterized by restriction enzyme analysis (REA). These isolates had previously been categorized into 19 PvuII ribotypes. Twenty restriction endonucleases were evaluated for use in REA. Digestion of chromosomal DNA with HinfI, followed by submarine electrophoresis in agarose gels and staining with ethidium bromide, produced DNA fragments in the 4.0- to 10-kb range, which readily discriminated B. bronchiseptica isolates, resulting in 48 fingerprint patterns. Moreover, AluI digestion of chromosomal DNA produced 39 distinct fingerprint profiles with DNA fragments ranging from 6.0 to 20.0 kb. While REA frequently provided more discriminatory power than ribotyping, there were examples where the use of ribotyping was more discriminatory than REA. Passage of selected isolates up to passage 25 did not change the REA profile. Moreover, the Bvg phase did not alter the fingerprint profile of chromosomal DNA from B. bronchiseptica strains digested with HinfI orAluI. Based on the results presented herein, the combination of REA and ribotyping should provide valuable information in understanding the molecular epidemiology of B. bronchiseptica infections.

scholarly journals Interaction of RecBCD Enzyme with DNA at Double-Strand Breaks Produced in UV-Irradiated Escherichia coli: Requirement for DNA End Processing

1998 ◽  
Vol 180 (21) ◽  
pp. 5639-5645 ◽  
Author(s):  
Brigitte Thoms ◽  
Wilfried Wackernagel
Keyword(s):  
Excision Repair ◽  
Uv Light ◽  
Single Strand ◽  
Duplex Dna ◽  
Dna Fragments ◽  
Chromosomal Dna ◽  
Phage T4 ◽  
Recbcd Enzyme ◽  
Dna Ends ◽  
Activity Decrease

ABSTRACT The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m2). The activity decrease was seen by an increase in survival of phage T42 − of about 200-fold (phage T42 − has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb +) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, andsbcCD, particularly in the recJ sbcCD andxonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.

scholarly journals Effects of mutation position on frequency of marker rescue by homologous recombination.

1992 ◽  
Vol 12 (8) ◽  
pp. 3609-3613 ◽  
Author(s):  
L Jiang ◽  
A Connor ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Normal Function ◽  
Constant Region ◽  
Dna Fragments ◽  
Wild Type ◽  
End Effect ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Marker Rescue ◽  
Immunoglobulin Mu

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

Sign in / Sign up

Export Citation Format

Share Document