Skip to main content
construction release_alert
Scholars@Duke will be undergoing maintenance April 11-15. Some features may be unavailable during this time.
cancel

Subtractive cloning: new genes for studying inflammatory disorders.

Publication ,  Journal Article
Rebrikov, D; Desai, S; Kogan, YN; Thornton, AM; Diatchenko, L
Published in: Ann Periodontol
December 2002

Understanding of the biology of interaction between pathogens and host is the central question in studying inflammatory disorders. Subtractive DNA cloning is one of the most efficient and comprehensive methods available for identifying eukaryotic genes regulated under specific physiological conditions, including inflammation and host response. Here we explore the utility of subtractive DNA cloning and describe suppression subtractive hybridization (SSH), a polymerase chain reaction (PCR)-based DNA subtraction method that has been developed and evolved in our labs over several years. The SSH method possesses a number of advantages as compared to other subtractive cloning techniques, making it one of the most adventitious methods for cloning differentially expressed genes. Besides isolation of differentially expressed eukaryotic mRNAs, subtractive DNA cloning can be used to identify genes that are differentially expressed between diverse bacterial species. These genes can be of great interest, as some may encode strain-specific traits such as drug resistance, or bacterial surface proteins involved in determining the virulence of a particular strain. Other genes may be useful as markers for epidemiological or evolutionary studies. To demonstrate the potential of the SSH technique, we describe here the comprehensive characterization of 2 SSH subtracted libraries constructed in our laboratories. One library was created using eukaryotic cDNA subtraction and is specific for mRNAs up-regulated in CD25 positive cells from mouse lymph nodes as compared to CD25 negative cells. The second subtracted library is specific for a methicillin-resistant Staphylococcus aureus bacterial strain, but not in a methicillin-sensitive strain. The bacterial genomes of these 2 strains have been completely sequenced and this second library provides an excellent reference for testing the ability of SSH to recover all strain-specific gene content. The analysis of these 2 subtracted libraries serves as the basis for a discussion of the strength and limitations of the SSH technique. We will also compare and contrast subtractive DNA cloning to other current technologies used to isolate differentially expressed genes.

Duke Scholars

Published In

Ann Periodontol

DOI

ISSN

1553-0841

Publication Date

December 2002

Volume

7

Issue

1

Start / End Page

17 / 28

Location

United States

Related Subject Headings

  • Staphylococcus aureus
  • Receptors, Interleukin-2
  • Polymerase Chain Reaction
  • Nucleic Acid Hybridization
  • Mice, Inbred BALB C
  • Mice
  • Methicillin Resistance
  • Inflammation
  • Genes, Bacterial
  • Gene Library
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Rebrikov, D., Desai, S., Kogan, Y. N., Thornton, A. M., & Diatchenko, L. (2002). Subtractive cloning: new genes for studying inflammatory disorders. Ann Periodontol, 7(1), 17–28. https://doi.org/10.1902/annals.2002.7.1.17
Rebrikov, Denis, Sejal Desai, Yakov N. Kogan, Angela M. Thornton, and Luda Diatchenko. “Subtractive cloning: new genes for studying inflammatory disorders.Ann Periodontol 7, no. 1 (December 2002): 17–28. https://doi.org/10.1902/annals.2002.7.1.17.
Rebrikov D, Desai S, Kogan YN, Thornton AM, Diatchenko L. Subtractive cloning: new genes for studying inflammatory disorders. Ann Periodontol. 2002 Dec;7(1):17–28.
Rebrikov, Denis, et al. “Subtractive cloning: new genes for studying inflammatory disorders.Ann Periodontol, vol. 7, no. 1, Dec. 2002, pp. 17–28. Pubmed, doi:10.1902/annals.2002.7.1.17.
Rebrikov D, Desai S, Kogan YN, Thornton AM, Diatchenko L. Subtractive cloning: new genes for studying inflammatory disorders. Ann Periodontol. 2002 Dec;7(1):17–28.

Published In

Ann Periodontol

DOI

ISSN

1553-0841

Publication Date

December 2002

Volume

7

Issue

1

Start / End Page

17 / 28

Location

United States

Related Subject Headings

  • Staphylococcus aureus
  • Receptors, Interleukin-2
  • Polymerase Chain Reaction
  • Nucleic Acid Hybridization
  • Mice, Inbred BALB C
  • Mice
  • Methicillin Resistance
  • Inflammation
  • Genes, Bacterial
  • Gene Library