Two-dimensional motion of DNA bands during 120° pulsed-field gel electrophoresis. I. Effect of molecular weight

Biopolymers ◽  
1995 ◽  
Vol 35 (3) ◽  
pp. 297-306 ◽  
Author(s):  
M. Shane Hutson ◽  
George Holzwarth ◽  
Thomas Duke ◽  
Jean-Louis Viovy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Two Dimensional ◽  
Pulsed Field

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

2006 ◽  
Vol 52 (9) ◽  
pp. 857-867 ◽  
Author(s):  
Ellen L Dally ◽  
Thereza S.L Barros ◽  
Yan Zhao ◽  
ShaoPing Lin ◽  
Bruce A Roe ◽  
...  
Keyword(s):  
Genetic Map ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Enzyme Analysis ◽  
Two Dimensional ◽  
Pulsed Field ◽  
Spiroplasma Kunkelii ◽  
Corn Stunt ◽  
Physical And Genetic Map

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.Key words: Spiroplasma kunkelii CR2-3x, corn stunt spiroplasma, mollicutes, genome mapping, two-dimensional pulsed-field gel electrophoresis.

scholarly journals Quantitation of Integrated HIV Provirus by Pulsed-Field Gel Electrophoresis and Droplet Digital PCR

2018 ◽  
Vol 56 (12) ◽  
Author(s):  
Steven M. Lada ◽  
Karissa Huang ◽  
D. Jake VanBelzen ◽  
Luis J. Montaner ◽  
Una O'Doherty ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Pulsed Field Gel Electrophoresis ◽  
Prolonged Treatment ◽  
High Molecular Weight Dna ◽  
Pulsed Field ◽  
Hiv Dna ◽  
Nonparametric Correlation

ABSTRACTWe utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIVgagsequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to theAlu-gagquantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated withAlu-gagqPCR results (r= 0.7052;P= 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

Two-dimensional pulsed-field gel electrophoresis of yeast chromosomes: evidence for triplex-mediated DNA condensation

1993 ◽  
Vol 71 (3-4) ◽  
pp. 190-196 ◽  
Author(s):  
Ken J. Hampel ◽  
Jeremy S. Lee
Keyword(s):  
Gel Electrophoresis ◽  
Chromosome Condensation ◽  
Two Dimensions ◽  
Pulsed Field Gel Electrophoresis ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Pulsed Field ◽  
Triplex Formation ◽  
Lauryl Sarcosine ◽  
Effects Of Ph

The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresis. The first dimension was run at pH 8.0 in a 1% agarose gel. In the second dimension the electrophoresis conditions were identical, except that the pH was lowered and ethidium, spermine, or ionic detergents were added. Any mobility changes between the two dimensions could be identified as a deviation from the diagonal. At pH 6.0 the mobility of the chromosomes increases severalfold, whereas at pH 4.5 none of the chromosomes move into the agarose gel. The pH-induced mobility changes were reversed by the addition of 2 μg/mL of ethidium or 1% lauryl sarcosine. Alternatively, spermine at 1 μM enhanced the pH-mediated mobility changes. Hysteresis was also evident, since upon lowering the pH to 4.5 and then running the gel at pH 7 the mobilities were decreased. These results are interpreted in terms of pH-mediated triplex formation which causes chromosome condensation and thus mobility shifts. The effects of pH are reversed by ethidium which destabilizes triplexes, but enhanced by spermine which favours triplex formation. Therefore, chromosomes may be capable of spontaneous condensation which is mediated by tertiary interactions between appropriate duplex DNA sequences.Key words: triplex DNA, pulsed-field gel electrophoresis, chromosome structure, DNA mobility, chromosome condensation.

scholarly journals Chromosomal composition of the genome in the monomorphic diplokaryotic microsporidium Paranosema grylli: analysis by two-dimensional pulsed-field gel electrophoresis

2005 ◽  
Vol 52 (1-2) ◽  
pp. 145-157 ◽  
Author(s):  
Elena Nassonova ◽  
Emmanuel Cornillot ◽  
Guy Metenier ◽  
Nina Agafonova ◽  
Boris Kudryavtsev ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Two Dimensional ◽  
Pulsed Field
Sign in / Sign up

Export Citation Format

Share Document