Please cite as: CSH Protocols; 2008; doi:10.1101/pdb.prot4923

This Protocol
Right arrow Full Text
Right arrow Update/discuss this protocolDiscussion icon
Right arrow Alert me when this protocol is cited
Right arrow Alert me when comments are published
Right arrow Alert me if a correction is posted
Services
Right arrow Similar protocols in this database
Right arrow Alert me to new releases of protocols
Right arrow Save to Personal Folders
Right arrow Download to citation manager
Right arrow Printer-friendly versionPrinter-friendly version
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arneson, N.
Right arrow Articles by Done, S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Arneson, N.
Right arrow Articles by Done, S.
Related Collections
Right arrow Molecular Biology, general
Right arrow DNA Sequencing
Right arrow Genomic DNA
Right arrow Libraries
Right arrow Libraries, general
Right arrow Genomic Libraries
Right arrow Genome Analysis
Right arrow Polymerase Chain Reaction (PCR)
Right arrow Polymerase Chain Reaction (PCR), general
Right arrow Amplification of DNA by PCR
Right arrow Bioinformatics/Genomics, general
Right arrow Genetics, general
Right arrow Genetic Variation
Right arrowRelated Protocols
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Scion Methods Express Manuals

protocolProtocol

Whole-Genome Amplification by Single-Cell Comparative Genomic Hybridization PCR (SCOMP)

Nona Arneson, Simon Hughes, Richard Houlston, and Susan Done

This protocol was adapted from "PCR-Based Whole Genome Amplification," Chapter 18, in PCR (eds. Hughes and Moody). Scion Publishing Ltd., Oxfordshire, UK, 2007.


INTRODUCTION

PCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Ligation-mediated PCR techniques involve ligating an adaptor sequence onto a "representation" of DNA molecules, generated following enzymatic digestion, random shearing, or chemical cleavage. Single-cell comparative genomic hybridization (SCOMP), described in this protocol, is a form of ligation-mediated PCR that was specifically designed for WGA of extremely limited sources of genomic DNA. The reaction volume is purposely kept to a minimum, and all buffers are optimized to eliminate the need to purify the reaction between steps. In addition, the entire reaction is performed in a single tube. This avoids initial template loss and reduces the risk of PCR contamination. SCOMP begins by converting the genome to a high-complexity representation with a fragment size of <2 kb by digesting with the restriction enzyme MseI. This results in a smear in the range of 100-1500 bp. Following enzyme digestion, adaptors containing specific primer sequences (specific to the restriction enzyme used) are ligated onto the ends of the genomic DNA and amplified in a high-stringency PCR. This results in a smear of PCR products in the range of 100-1500 bp, which can be visualized by agarose gel electrophoresis.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?

Related Protocols

Agarose Gel Electrophoresis
Joseph Sambrook and David W. Russell
CSH Protocols 2006: 4020. [Extract] [Full Text]

Whole-Genome Amplification by Degenerate Oligonucleotide Primed PCR (DOP-PCR)
Nona Arneson, Simon Hughes, Richard Houlston, and Susan Done
CSH Protocols 2008: 4919. [Abstract] [Full Text]

GenomePlex Whole-Genome Amplification
Nona Arneson, Simon Hughes, Richard Houlston, and Susan Done
CSH Protocols 2008: 4920. [Abstract] [Full Text]

Whole-Genome Amplification by Improved Primer Extension Preamplification PCR (I-PEP-PCR)
Nona Arneson, Simon Hughes, Richard Houlston, and Susan Done
CSH Protocols 2008: 4921. [Abstract] [Full Text]

Whole-Genome Amplification by Adaptor-Ligation PCR of Randomly Sheared Genomic DNA (PRSG)
Nona Arneson, Simon Hughes, Richard Houlston, and Susan Done
CSH Protocols 2008: 4922. [Abstract] [Full Text]