PCR & Sequencing

PCR & Sequencing

The Polymerase Chain Reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy and cheap tool to amplify a focused segment of DNA, useful in the diagnosis and monitoring of genetic diseases, identification of criminals (under the field of forensics), and studying the function of a targeted segment.

The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, which the method is named after, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. PCR is the method of choice in preparing reactions for NGS (Next Generation Sequencing).

MagSi-NGSPREP provides a convenient tool for ultra-fast and efficient purification and size selection of DNA products. Magnetic bead-based MagSi-NGSPREP Plus offers an efficient solution for both size selection and clean-up of the successive enzymatic reactions in library preparation for NGS applications.

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. So far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as pyrosequencing are gaining an increasing share of the sequencing market. More genome data are now being produced by pyrosequencing than Sanger DNA sequencing. 

An alternative to the labelling of the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the incorporation of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability. This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper.

Magnetic bead based MagSi-DNA cleanFIX offers one single product for efficient PCR clean-up and Dye terminator removal from DNA Sanger sequencing reactions, enabling researchers to address various genomic clean-up steps with the same product. Magnetic bead-based MagSi-DT Removal offers an efficient solution for Dye-Terminator removal from BigDye® sequencing reactions.