PCR & Sanger Sequencing

Polymerase Chain Reaction

PCR is shorthand for a simple but very useful procedure in molecular biology called the polymerase chain reaction. It is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies. Polymerase chain reaction (PCR) is an efficient and cost-effective way to copy or “amplify” small segments of DNA or RNA. Using PCR, millions of copies of a section of DNA are made in just a few hours, yielding enough DNA required for analysis. This innovative yet simple method allows clinicians to diagnose and monitor diseases using a minimal amount of sample, such as blood or tissue. It is an indispensable tool in modern molecular biology and has transformed scientific research and diagnostic medicine. Some of the myriad of applications of the PCR technique include the following:

• Disease diagnosis.
• Because a specific sequence can be amplified greatly, much less clinical material is needed to make a diagnosis.
• PCR can be used to detect pathogens that are difficult to culture, such as the causative agents for Lyme disease or for AIDS.
• PCR can be used for cancer diagnosis.

Sanger sequencing

DNA sequencing is a laboratory method used to determine the sequence of a DNA molecule. The method was developed by Frederick Sanger in 1975, who was later awarded the Nobel Prize in chemistry in 1980 for his contributions to understanding DNA sequences. Consequently, it is often referred to as Sanger Sequencing. Sanger sequencing is a targeted sequencing technique that uses oligonucleotide primers to seek out specific DNA regions. Sanger sequencing is a robust testing strategy able to determine whether a point mutation or small deletion/duplication is present. It has been widely used for several decades in many settings, including defining the mutational spectrum of a tumor as well as identifying a constitutional variant in diagnostic testing. Primers can be created to cover several regions (amplicons) to cover any size region of interest.