Guide to DNA Amplification

Imagine, for a moment, looking for a needle in a haystack—a single gene or sequence of interest in the haystack that is the genome. DNA amplification allows researchers to multiply that needle by a million- or billion-fold, making it possible to detect and study. Because of its versatility and flexibility, DNA amplification is used in research, diagnostics, environmental science, and forensic investigation, with downstream applications including molecular cloning, sequencing, and genome-wide studies. This article will discuss modern methods of DNA amplification, including polymerase chain reaction (PCR), whole genome amplification, and isothermal DNA amplification methods.

PCR and the genomics revolution

PCR revolutionized research in genetics and genomics by allowing specific parts of DNA to be amplified in vitro for the first time. Nearly 40 years after its initial publication, PCR is still the workhorse technique in molecular biology due to its low cost, ease of use, and flexibility.

The concept behind PCR is straightforward: mix DNA with oligonucleotide primers, free nucleotides, ATP, salts, DNA polymerase, and cycle through denaturation, annealing, and extension phases. By going through 25 to 35 cycles, PCR exponentially generates more copies of the target sequence.

Conventional PCR

The most common application of PCR, conventional PCR produces an amplicon that can be visualized on an agarose gel. Endpoint PCR is generally not quantitative, where the presence or absence of a band gives a simple yes/no answer to the presence of the sequence of interest. This technique can be used as an endpoint—as is the case in genotyping animals or asking whether a certain microbe is present in a sample—or it can be used in downstream applications such as molecular cloning or Sanger sequencing.

An advantage of conventional PCR is the flexibility of its design: it can be used in simple or multiplexed designs (using a single pair of primers to produce one amplicon or multiple primer pairs of primers to simultaneously probe more than one target). Samples for conventional PCR can be purified DNA, bacterial colonies (colony PCR), or supernatant from digested tissue—commonly used for genotyping animals. Limitations of conventional PCR are that the results are not quantitative and that the amplication by itself is not sensitive enough to detect small mutations; however, sequencing an amplicon can give insight into mutation status, augmenting PCR’s flexibility.

Quantitative PCR

Quantitative PCR (qPCR), also known as real-time PCR, uses fluorescent dyes or labeled probes to analyze the abundance of the PCR product in real-time. This method allows for quantitative experiments versus the binary answer that endpoint PCR gives. As such, qPCR is commonly used in gene expression studies, where RNA is reverse-transcribed into complementary DNA (cDNA) in reverse transcription qPCR (RT-qPCR). Like conventional PCR, qPCR can also be done in simple or multiplex designs.

Also see the Guide to Selecting a PCR Method for more information on the most common PCR methods, as well as their benefits and applications.

Whole genome amplification

Using oligonucleotide primers and a polymerase in vitro extends beyond PCR. Whole genome amplification takes the PCR concept but scales it up in a massively parallel way to reproduce entire genomes regardless of sequence. Whole genome amplification requires high-quality source material and low bias. Regions of the genome with unusually low or high GC content may produce regions of the genome that are over- or under-represented in the amplified genome.

Hybrid capture amplification

Hybrid capture is a targeted amplification technique that uses oligonucleotide probes, also known as baits, to “capture” target sequences of interest. The probes can either be physically anchored to a substrate such as a microarray or be biotinylated for later purification. Hybrid capture is effective for analyzing a high number of genes of interest or whole exomes and has been used for whole genome amplification. Limitations of hybrid capture include high cost, long preparation time for oligonucleotide probes, and high level of required expertise to create and analyze the amplified libraries.

Multiple displacement amplification (MDA)

MDA is one of the most used genome-wide amplification techniques, using the high-fidelity Phi29 polymerase and random hexamers. The Phi29 polymerase has proofreading capability for high fidelity replication and can produce amplicons up to 100 kb in length. This technique can amplify entire genomes from minute amounts of DNA, producing 1–2 µg of high-quality DNA from a single cell, which can be used in sequencing, microarrays, and other analyses.

Isothermal amplification techniques

A limitation of PCR is the need for thermal cycling. Isothermal DNA amplification techniques work at a constant temperature, making them ideal for field or point-of-care applications.

Loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) uses 4–6 designed primers and a polymerase with strand-displacement capacity. These primer sets amplify the DNA at a constant 65°C, requiring only the primers, master mix, DNA sample, and a heat block or water bath for temperature control. The products of the LAMP reaction can be visualized by gel electrophoresis or with indicator dyes like hydroxynaphthol blue or SYBR green, making them well-suited for point-of-care or in-the-field uses in medicine and agriculture.

Rolling circle amplification

This technique uses an exonuclease-capable polymerase to copy a circularized piece of DNA. The polymerase processes along the circular DNA, making several circuits and producing multiple repeats of the template sequence. Rolling circle amplification is unique in that it can be used to generate functionally active nucleic acids for use in biosensors for diagnostic applications.

With the refinement of PCR and advent of new technologies, the opportunities to find and characterize needles in the genomic haystack have never been brighter.