Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column.
Analysis and purification of specific PCR products from PCR reactions can be problematic due to several issues relating to amplification and low product yield. The use of HPLC as a preparative tool in PCR product analysis is common but has not replaced traditional electrophoretic techniques for purifying DNA to be used in subsequent experiments. Gel purification of PCR products can result in a net loss greater than 50% of the starting DNA amount. Thus, this method of recovery can become the limiting factor in the overall cloning protocol. This paper describes a simple and relatively inexpensive micro-preparative HPLC method to purify and analyze nM quantities of DNA. A microbore polyethyleneimine-based anion-exchange column fractionates PCR mixtures in less than 40 min with a recovery of the purified specific product as high as 80%, thus eliminating the need for gel purification. Using this method, nested PCR products from Sindbis virus differing by 18 bp in some cases and a 277 bp fragment from West Nile virus were resolved and quantified. This method differs from existing methodologies because separation is based on size and charge as well as the overall G + C content of the PCR product.
Department of Molecular and Structural Biochemistry, North Carolina State University, Campus Box 7622, Raleigh, NC 27695 7622, USA. raquel_hernandez@ncsu.edu
Raquel
R
Hernandez
Steevenson
S
Nelson
Jeffery R
JR
Salm
Dennis T
DT
Brown
Andrew J
AJ
Alpert
D000838:Anions / Q000032:analysis / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012845:Sindbis Virus / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors; D014773:Virology / Q000379:methods; D014902:West Nile virus / Q000235:genetics / Q000302:isolation & purification*
D000838:Anions; D004279:DNA, Viral