Bench Marks » Polymerase Chain Reaction (PCR)

Zinc Finger Nuclease Deletions

David Crotty — Mon, 02 Aug 2010 14:55:08 +0000

Zinc finger nucleases (ZFNs) are artificial restriction enzymes made by fusing an engineered zinc finger DNA-binding domain to the DNA cleavage domain of a restriction enzyme. ZFNs can be used to generate targeted genomic deletions of large segments of DNA in a wide variety of cell types and organisms. In the August issue of Cold Spring Harbor Protocols, Jin-Soo Kim and colleagues present Analysis of Targeted Chromosomal Deletions Induced by Zinc Finger Nucleases, a detailed protocol for the detection and analysis of large genomic deletions in cultured cells introduced by the expression of ZFNs. The method described allows researchers to detect and estimate the frequency of ZFN-induced genomic deletions by simple PCR-based methods. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

DNA Methylation Profiling

David Crotty — Tue, 08 Dec 2009 01:51:50 +0000

With the recent progress in understanding epigenetic mechanisms, methods for profiling patterns of DNA modification have become important tools for analysis of gene regulation. DNA methylation, in which cytosine is modified to form 5-methylcytosine, is a well-characterized epigenetic modification essential for normal development in plants and mammals. In the December issue of Cold Spring Harbor Protocols, Jon Reinders presents Amplification of Bisulfite-Converted DNA for Genome-Wide DNA Methylation Profiling. This method utilizes the treatment of DNA with sodium bisulfite, which converts unmethylated cytosine to uracil (5-methylcytosine is not converted). This is followed with PCR amplification, where the uracil amplifies as thymine, creating a C-to-T transition. The genome can then be analyzed for these transitions using an array-based platform. Reinders protocol mitigates the major issues with bisulfite conversion (DNA fragmentation and poor reproducibility) and reduces bias during the amplification step. While the protocol is optimized for use in Arabidopsis, it can potentially be adapted for use in other organisms.

Nested Patch PCR

David Crotty — Mon, 13 Jul 2009 14:02:39 +0000

Nested Patch PCR is a method designed to identify SNPs and mutations across many targeted loci for many samples in parallel. In the July issue of Cold Spring Harbor Protocols, Robi Mitra and colleagues from Washington University present Nested Patch PCR for Highly Multiplexed Amplification of Genomic Loci, a method where a large number (greater than 90) of targeted loci from genomic DNA are simultaneously amplified in the same reaction. These amplified loci can then be sequenced on a second-generation sequencing machine to detect single nucleotide polymorphisms (SNPs) and mutations.

Methods that employ mulitplexing during PCR reactions are often hampered by increased interprimer interactions that inhibit uniform amplification and increased formation of mispriming products. The protocol presented here was designed to reduce these two problems and results in a high specificity, with 90% of sequencing reads mapping to targeted loci. Nested Patch PCR is well-suited for the amplification of an intermediate number (100-1000) of targeted regions across a large number of samples and it offers a simple workflow that is compatible with 96-well plates and sample-specific DNA barcodes.

PCR Primers

David Crotty — Fri, 10 Apr 2009 17:37:56 +0000

One of the reasons Cold Spring Harbor Protocols exists is to allow us to keep valuable information available after the books where it was published go out of print. One of April’s featured articles, Optimization and Troubleshooting in PCR, is a great example of this.

Our PCR Primer manual has gone out of print, but the contents are still relevant and important to researchers in many labs. PCR is often difficult to optimize, and failure to do so can lead to undefined and unwanted products, or a complete lack of amplification altogether. To help avoid these issues, Kenneth Roux from Florida State University wrote the chapter that has been adapted for publication here. The article addresses various optimization strategies including touchdown PCR and hot-start PCR. Magnesium concentration, buffer pH, and cycling conditions are also considered. Like all our featured articles, Optimization and Troubleshooting in PCR is freely accessible to subscribers and non-subscribers alike.

This joins our collection of other valuable articles from PCR Primer like PCR Primer Design, Strategies for Overcoming PCR Inhibition, and Setting Up a PCR Laboratory. A complete listing of our PCR-related articles can be found here.

Biofilm Analysis

David Crotty — Mon, 13 Oct 2008 22:47:51 +0000

Biofilms are the natural state of an estimated 99% of prokaryotes in the environment and are defined as an aggregation of microorganisms in a self-created matrix on a surface. Examples are plaque on teeth, or the slime on rocks at the bottom of a river. Because these biofilms can’t be effectively cultured in the laboratory, new techniques are being developed to isolate material from the environment, allowing for a better understanding of the microbes responsible for many diseases and infections. The field of metagenomics, “the culture-independent analysis of a mixture of microbial genomes (termed the metagenome) using an approach based either on expression or on sequencing” is rapidly growing. October’s issue of CSH Protocols presents two useful methods for the study of biofilms from the laboratory of Michael J. Franklin, of Montana State University’s Center for Biofilm Engineering.

Isolation of RNA and DNA from Biofilm Samples Obtained by Laser Capture Microdissection Microscopy describes techniques for embedding biofilms in cryoembedding resin, producing thin sections and isolating discrete sections through laser capture microdissection microscopy. RNA or DNA is then extracted from these discrete populations of cells.

qRT-PCR of Microbial Biofilms takes the RNA isolated in the first method and allows analysis of the number of RNA transcripts of specific genes from bacteria growing in biofilms through quantitative reverse transcriptase real time PCR.