Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens.
We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target ('tester'). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens ('driver'). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies.
Ganova-Raeva
Lilia
L
Centers for Disease Control and Prevention, National Center for Infectious Diseases, Division of Viral Hepatitis/Laboratory Branch, Atlanta, GA 30329, USA. lkg7@cdc.gov
Zhang
Xinjian
X
Cao
Fengli
F
Fields
Howard
H
Khudyakov
Yury
Y
D002139:Caliciviridae / Q000235:genetics / Q000302:isolation & purification; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D017931:DNA Primers; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D013698:Templates, Genetic
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012313:RNA; D004247:DNA