PCR Fragments

Polymerase chain reaction (PCR) kick started molecular biology and has become a staple procedure across biological research. Developed in the early 1980s, PCR provided biologists and biochemists the means to selectively amplify desired regions of DNA for further study.

AATI PCR Fragments Photo

What is PCR?

Since its inception nearly 40 years ago, PCR has evolved numerous subtypes to fit different experimental needs. While there are now many subtypes of PCR, all have the same basic requirements: template (often plasmid or genomic DNA), DNA primers, a thermophilic polymerase, and a thermocycler to modulate reaction temperature. Every type of successful PCR ends with the generation of one or more amplicons (or PCR fragments), for use in subsequent analysis or downstream applications.

Complete analysis of PCR amplicons requires the implementation of a protocol capable of delivering reliable sizing, quantification, and qualification. AATI produces a variety of solutions for the complete and accurate analysis of PCR fragments across a broad range of amplicon lengths.

PCR is ubiquitous in life sciences research, from ecology laboratories to cancer research groups, it is a valuable tool enabling discovery at every level. PCR has enabled or simplified various molecular biology workflows, including genetic engineering, molecular cloning, genotyping, and next-generation sequencing, among others.

The polymerase chain reaction (PCR) is a pervasive tool across biological research, from ecology to cancer genetics. The proper analysis of PCR products requires a method of sufficient sensitivity and exceptional resolution. However, PCR fragments are commonly analyzed by agarose gel electrophoresis, which, while inexpensive and readily available in most laboratories, requires a large amount of sample and has poor separation resolution. Furthermore, agarose gel electrophoresis does not scale to effectively meet the needs of high-throughput environments.

Accelerate PCR fragment analysis with the instrument offerings of Agilent - formerly Advanced Analytical (AATI). Parallel capillary electrophoresis provides fast, reliable, and accurate analysis of DNA fragments. Four instruments are available from Agilent - formerly AATI that are well-suited to PCR amplicon analysis:

A variety of reagent kits are available for these instruments suited to the qualitative analysis of PCR fragments as well as for more specialized applications, such as the detection of genetic markers.

Qualitative PCR Fragment Analysis

In most cases, reagent kits are selected based on the size of the PCR fragments of interest and the resolution required. However certain kits confer additional benefits including dealing with challenging samples and faster run times. Difficulties in PCR fragment analysis commonly arise due to sequence composition and structural conformation, leading to separation artifacts such as shadow peaks and size shifts.

Figure 1. Separation of a challenging PCR fragment using the DNF-905 dsDNA 905 Reagent Kit. The DNF-905 dsDNA 905 Reagent Kit using a specially formulated separation gel to ameliorate challenges that arise due to conformation and sequence composition, including shadow peaks and size shifts. Capillary electrophoresis performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55).

Genetic Marker Detection

A common application of PCR is basic genotyping and the determination of the presence or absence of specific genetic markers, frequently microsatellites. Important to forensics, population genetics, and medical research, microsatellites consist of short (2 bp – 5 bp) sequences repeated up to 50 times. With 3 bp resolution, the Fragment Analyzer, 5400 Fragment Analyzer, and the ZAG DNA Analyzer System provide the requisite resolution for the reliable and accurate analysis of microsatellites and other genetic markers.

Figure 2. Separation of two microsatellite peaks with the DNF-935 dsDNA 935 Reagent Kit. The 3 bp resolution provided by the capillary electrophoresis instruments from Agilent - formerly AATI enables the confident analysis of closely sized PCR fragments. Capillary electrophoresis performed using a Fragment Analyzer equipped with a Short Capillary Array (33-55).

PCR Fragment Analysis Kits

There are a variety of kits for the Fragment Analyzer, 5400 Fragment Analyzer, and the ZAG DNA Analyzer System. The Fragment Analyzer instruments are versatile, capable of more than PCR fragment analysis, while the ZAG was specially designed to excel at high-throughput PCR fragment analysis. However, all instruments deliver down to 3 bp resolution and reliable relative quantification of DNA fragments. Across available reagent kits, the sizing range for each instrument covers 1 bp – 20,000 bp.

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