The preparation of high-molecular-weight DNA from rice and its analysis by pulsed-field gel electrophoresis

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.

Download Full-text

Analysis of high-molecular-weight free nuclear DNAs revealed in mammalian cells by pulsed-field gel electrophoresis

Molecular Biology ◽

10.1007/bf02759659 ◽

2000 ◽

Vol 34 (3) ◽

pp. 313-320

Author(s):

A. V. Budilov ◽

D. A. Domninskii ◽

V. I. Popenko ◽

T. I. Sukhova ◽

I. V. Botezatu ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Gel Electrophoresis ◽

Mammalian Cells ◽

Pulsed Field Gel Electrophoresis ◽

Pulsed Field

Download Full-text

Somatic chromosome karyotype of tomato based on in situ hybridization of the TGRI satellite repeat

Genome ◽

10.1139/g89-543 ◽

1989 ◽

Vol 32 (6) ◽

pp. 992-998 ◽

Cited By ~ 54

Author(s):

Nora L. V. Lapitan ◽

Martin W. Ganal ◽

Steven D. Tanksley

Keyword(s):

Molecular Weight ◽

In Situ Hybridization ◽

Satellite Dna ◽

Gel Electrophoresis ◽

Morphological Characteristics ◽

Pulsed Field Gel Electrophoresis ◽

Mitotic Chromosomes ◽

High Molecular Weight Dna ◽

Pulsed Field

A karyotype of tomato mitotic chromosomes was constructed based on in situ hybridization to a 162-bp telomeric DNA repeat, TGRI. Variation in the spatial and quantitative distribution of this repeat creates distinct patterns for most of the chromosomes, which along with other morphological characteristics (i.e., length and arm length ratio), allow the identification of each of the 12 mitotic chromosomes of tomato. The structure and physical size of the TGRI clusters were further investigated by means of pulsed-field gel electrophoresis. Approximately 30 hybridizing fragments were observed in the range of 25 to 1000 kb when high molecular weight DNA was digested with BglII and probed with TGRI. The total molecular weight of these fragments is approximately 14 million bp, which is close to the estimated total length of TGRI in the genome (12.5 million bp) based on genomic reconstruction experiments. The results suggest that most of the TGRI clusters consist of single, uninterrupted blocks of satellite DNA. Assignment of somatic chromosomes, identified by TGRI hybridization to the previously established tomato linkage groups, was accomplished via in situ hybridization to mitotic spreads of primary trisomic lines. Using this information, we estimate the somatic length and DNA content of each of the tomato chromosomes and chromosome arms. Key words: Lycopersicon esculentum, somatic karyotype, in situ hybridization, satellite DNA, pulsed-field gel electrophoresis.

Download Full-text

Preparation of high molecular weight plant DNA and analysis by pulsed field gel electrophoresis

Plant Molecular Biology Manual ◽

10.1007/978-94-011-0511-8_26 ◽

1994 ◽

pp. 401-421 ◽

Cited By ~ 1

Author(s):

Raymond A. J. J. Van Daelen ◽

Pim Zabel

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Gel Electrophoresis ◽

Pulsed Field Gel Electrophoresis ◽

Pulsed Field ◽

Plant Dna

Download Full-text

Construction of a MluI-YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions

Genome ◽

10.1139/g97-116 ◽

1997 ◽

Vol 40 (6) ◽

pp. 896-902 ◽

Cited By ~ 12

Author(s):

Michael Kleine ◽

Christian Jung ◽

Wolfgang Michalek ◽

Thomas Diefenthal ◽

Harald Dargatz

Keyword(s):

Hordeum Vulgare ◽

Gel Electrophoresis ◽

Yeast Artificial Chromosome ◽

Single Copy ◽

Pulsed Field Gel Electrophoresis ◽

Hordeum Vulgare L ◽

High Molecular Weight Dna ◽

Pulsed Field ◽

Artificial Chromosomes ◽

Yac Library

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10–30% of the colonies were recombinant, as visualized by red–white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE–1 copia–like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector–insert junctions.Key words: yeast artificial chromosomes, YAC, Hordeum vulgare, pulsed-field gel electrophoresis.

Download Full-text