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.

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

Genome ◽  
1989 ◽  
Vol 32 (6) ◽  
pp. 992-998 ◽  
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.

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

Genome ◽  
1997 ◽  
Vol 40 (6) ◽  
pp. 896-902 ◽  
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.

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

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

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

Pulsed-field gel electrophoresis analysis of the phaseolin locus region in Phaseolus vulgaris

Genome ◽  
1996 ◽  
Vol 39 (4) ◽  
pp. 722-729 ◽  
Author(s):  
Víctor Llaca ◽  
Paul Gepts
Keyword(s):  
Phaseolus Vulgaris ◽  
Multigene Family ◽  
Gel Electrophoresis ◽  
Genomic Region ◽  
Pulsed Field Gel Electrophoresis ◽  
Gene Arrangement ◽  
Pcr Analysis ◽  
Range Restriction ◽  
High Molecular Weight Dna ◽  
Pulsed Field

Phaseolin is the major seed storage protein of common bean (Phaseolus vulgaris L.). It is encoded by a small multigene family of 6–9 genes that are clustered in a single complex locus (Phs). We have constructed a long-range restriction map of the phaseolin genomic region, including the Phs locus and two flanking marker loci, D1861 and Bng060. Using a combination of high molecular weight DNA isolation, one- and two-dimensional pulsed-field gel electrophoresis of single and double restriction digests followed by Southern hybridization, and PCR analysis of individual fragments, we found that: (i) the maximum size of the Phs locus is 190 kb, (ii) the Phs locus may have increased in size during the evolution of P. vulgaris, (iii) the genomic region marked by D1861–Phs–Bng060 spans 5 cM, which corresponds to a maximum of 1.9 Mb, and (iv) the Phs locus could be oriented with respect to the two adjacent markers. Further progress in determining the gene arrangement in the Phs locus will require cloning and analysis of large DNA fragments containing phaseolin genes via BAC libraries. Key words : multigene family, physical distance, genome mapping, seed protein.

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