Co-Amplification+at+Lower+Denaturation+Temperature+–+Polymerase+Chain+Reaction+(COLD-PCR)

= = =__ Co-Amplification at Lower Denaturation Temperature - Polymerase Chain Reaction __=


 * Basic Description **

Edited by, Brian Senajor


 * Co-Amplification at Lower Denaturation Temperature – Polymerase Chain Reaction (COLD-PCR) **


 * Basic Description **

Co-amplification at lower denaturation temperature – polymerase chain reaction or COLD-PCR is a modified polymerase. The goal of COLD-PCR is to amplify certain alleles from a mixture of alleles. This differs from a regular PCR because a regular PCR amplifies all alleles present with no preference given to certain ones. This allows for easier detection of low level mutations in a genome. The principle behind COLD-PCR is that single nucleotide mismatches slightly alter the melting temperature of double-stranded DNA. This technique is similar to that of PCR except for the temperatures used. The first stage is the denaturation stage, which separates the double stands into single strands. Next the intermediate annealing stage is used to allow the hybridization between the mutant and the wild type of an allele to anneal to one another forming heteroduplexes. Next, the melting stage and since the heteroduplexes melt at lower temperatures they are denatured at the critical temperature instead of the melting temperature. This causes the mutant strands to be replicated while the normal strands are not replicated because they do not melt. The next stage is the primer annealing stage where primers will anneal to the single strands of DNA present. The last is the extension stage. The extension stage is performed with DNA polymerase. DNA polymerase extends the primer into acomplimentary strand of DNA to the template [1].


 * Purpose **

The purpose of this technique is to amplify a low level mutation that is not present in a large amount of a genome’s alleles. A COLD-PCR is able to selectively amplify these mutated strands and separate them out of the rest of the alleles so that they can be studied. This is important because a normal PCR will not selectively amplify, and instead amplifies everything equally. This would make it very difficult to study a mutated sequence.

**Origins**

This technique was first described by Li et al.in a Nature Medicine paper in 2008. This procedure has been used to in multiple proof-of-principle experiments and has just recently been commercialized in order to be used in more areas of research. Commercially it will be able to be used to rapidly detect low-level somatic DNA mutations.


 * Current Research **

Zhuang Zuo et al., authors of “Application of COLD-PCR for improved detection of KRAS mutations in clinical samples” used COLD-PCR in order to detect KRAS mutations. KRAS is a member of the RAS oncogene family which is important in cell signaling. Mutations in the KRAS gene have been found to lead to cancer. This study used both a COLD-PCR as well as a standard PCR. It was found that the COLD-PCR was able to amplify the mutant strands at an amount 4-fold greater than a standard PCR. This is important to the study because the authors wanted to be able to focus on strands with a mutated KRAS gene and it is much easier to be able to isolate them during replication instead of amplifying everything [2].

A second paper written by Irene Mancini et al., studies the KRAS mutations as well as BRAF mutations and how they relate to colorectal cancer. In this study the authors state that they got much better results when using a COLD-PCR than a regular PCR when amplifying the desired genes. They were able to confirm the presence of mutations using COLD-PCR and they also discovered that they were not able to determine this when using a regular PCR. This is important to the study because without using a COLD-PCR they would not have been able to obtain any worthwhile results [3].

A paper by George Santis et al., titled “Screening for EGFR and KRAS Mutations in Endobronchial Ultrasound Derived Transbronchial Needle Aspirates in Non-Small Cell Lung Cancer Using COLD-PCR” used a COLD-PCR to look for genetic abnormalities in cancer patients and then compared their findings to other sequencing techniques. They found that they were able to correctly sequence with every technique. However, a COLD-PCR gave a more defined peak when sequenced and made it easier to determine where a mutation has occurred. This is important because when determine a DNA sequence it can be difficult to differentiate between the peaks and since a COLD-PCR can make this easier it is a beneficial technique to employ [4].

The COLD-PCR technique is being used primarily in the field of cancer research currently. This is because it is able to easily and specifically amplify mutated strands of DNA that could potentially lead to cancer.


 * References **

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter (2008). Molecular Biology of The Cell. Garland Science Taylor & Francis Group, New York. [1] Li, J., Wang, L., Mamon, H., Kulke, M.H., Berbeco, R., and Makrigiorgos, G.M. 2008. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nature Medicine 14, 579–584. [2] Zhuang Zuo, Su S Chen, Pranil K Chandra, John M Galbincea, Matthew Soape, Steven Doan, Bedia A Barkoh, Hartmut Koeppen, L Jeffrey Medeirosand Rajyalakshmi Luthra. 2009. Application of COLD-PCR for improved detection of KRAS mutations in clinical samples. Modern Pathology 22, 1023-1031. [3] Irene Mancini, Claudio Santucci, Roberta Sestini, Lisa Simi, Nicola Pratesi, Fabio Cianchi, Rosa Valanzano, Pamela Pinzani, Claudio Orlando, The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer. The Journal of Molecular Diagnostics. 12.5, 705-711. [4] Santis G, Angell R, Nickless G, Quinn A, Herbert A, et al. 2011. Screening for EGFR and KRAS Mutations in Endobronchial Ultrasound Derived Transbronchial Needle Aspirates in Non-Small Cell Lung Cancer Using COLD-PCR. PLoS ONE 6(9).