Elsevier

Genomics

Volume 13, Issue 4, August 1992, Pages 1322-1324
Genomics

Short communication
A method for the rapid sequence-independent amplification of microdissected chromosomal material

https://doi.org/10.1016/0888-7543(92)90057-YGet rights and content

Abstract

We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3′ end and a defined sequence at its 5′ end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.

References (7)

  • S.K. Bohlander et al.

    A method for the rapid sequence-independent amplification of microdissected chromosomal material

    Am. J. Hum. Genet. Suppl

    (1991)
  • F.-T. Kao et al.

    Chromosome microdissection and cloning in human genome and genetic disease analysis

  • H.-J. Lüdecke et al.

    Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification

    Nature

    (1989)
There are more references available in the full text version of this article.

Cited by (167)

  • A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria

    2015, Journal of Microbiological Methods
    Citation Excerpt :

    This is illustrated by a high overall test specificity of 95% [TN/(TN + FP)] and a high predictive value of a positive test of 93% [TP/(TP + FP)] but also by a high overall test sensitivity of 97% [TP/(TP + FN)] and a high predictive value of a negative test of 98% [TN/(TN + FN)] (Table 2). Our novel DNA amplification and labeling system combines the advantages of the protocol of Bohlander et al. (1992) with potentially shorter working time and less expensive consumables but with use of the same optical readout. The adaptation of the microarray layout with additional antibiotic resistance genes is an ongoing process due to the continuous discovery of novel antibiotic resistance determinants.

  • Optimization of virus detection in cells using massively parallel sequencing

    2014, Biologicals
    Citation Excerpt :

    We found that DOP-PCR was sensitive for detecting small quantities of isolated viral nucleic acids (Fig. 1), when PCR products were evaluated by gel electrophoresis with Sanger sequencing, or by MPS, consistent with early experiments comparing DOP-PCR with other methods that showed a detection limit of 100–1000 copies [9]. Other nonspecific amplification techniques have been described in the literature, each previously used to identify viruses from clinical samples or in cell substrates and reagents [4,18–20,22–25]. The DOP-PCR primer differs from the degenerate primers used in other methods due to its specific 3′ anchor sequence.

  • Listeriosis in game birds

    2021, The Encyclopedia of Bacteriology Research Developments
View all citing articles on Scopus
View full text