Protocols for PCR may vary depending upon the applications. This video provides an overview on routine PCR, hot-start PCR, high-fidelity PCR, GC-rich PCR, and long PCR.
Variations in gene expression among cell types, tissues, and organisms at a specific time point are commonly examined by PCR. In this process, RNA is isolated from samples of interest and the messenger RNA (mRNA) is reverse-transcribed into complementary DNA (cDNA). The original levels of mRNA can then be determined from the quantity of cDNA amplified in PCR. This process is also known as reverse transcription PCR, or RT-PCR (Figure 1).
Figure 1. RT-PCR. RNA is reverse-transcribed into cDNA, which in turn is amplified by PCR (RT = reverse transcription, RTase = reverse transcriptase).
Endpoint PCR may be performed to quantitate RNA expression from the intensity of amplified products in a gel, although this is a semiquantitative approach. For example, input cDNA is serially diluted and then amplified. The endpoint yields of the various inputs are visualized on a gel (Figure 2), and the band intensities are then quantitated and normalized to those of a housekeeping gene reference to estimate the relative expression level of the amplified target [1,2]. Today, real-time PCR or qPCR has mostly replaced endpoint PCR to obtain more reliable and quantitative results of gene expression (learn more in quantitative PCR and reverse transcription).
Figure 2. PCR yields obtained from serial dilutions of input cDNA, visualized by staining of PCR products on an agarose gel.
PCR can be used to detect sequence variations in alleles in specific cells or organisms. An example is genotyping of transgenic organisms such as knock-out and knock-in mice . The primer sets are designed to flank regions of interest and assess genetic variations based on the presence or absence of an amplicon and/or its length (Figure 3). (App note: Mouse genotyping)
Figure 3. PCR for allelic genotyping of transgenic organisms. (A) Wildtype or transgenic sequences at a genomic locus can be detected by designing PCR primers specific to the regions of interest. (B) PCR products can be used to determine genotypes, as shown in this gel picture. Genomic DNA of wildtype (+/+) and transgenic (+/– and –/–) mice was used in these experiments.
To detect specific nucleotide mutations, however, the amplified sequences must be further analyzed. For instance, sequencing of PCR amplicons is one approach for studying single-nucleotide variations (SNVs) and single-nucleotide polymorphisms (SNPs). High-fidelity DNA polymerases are strongly recommended to prevent introduction of unwanted mutations during PCR.
Genotyping by PCR is also a fundamental aspect of genetic analyses of mutations in cancer and heredity.
PCR is widely used in cloning DNA fragments of interest, in a technique known as PCR cloning. In direct PCR cloning, the desired region of a DNA source (e.g., gDNA, cDNA, plasmid DNA) is amplified and inserted into specially designed compatible vectors. Alternatively, primers may be designed with additional nucleotides at their 5′ end for further manipulation before insertion. Examples of these add-on sequences include restriction sites for cloning via restriction digestion and ligation, vector-compatible sequences for ligation-independent cloning, and recombination sequences for multi-fragment assembly (Figure 4). (Learn more in the PCR cloning webinar)
Figure 4. PCR cloning: inserts prepared by PCR cloned into compatible vectors. (A) Direct PCR cloning techniques include TA and blunt-end cloning. (B) With indirect PCR cloning, amplicons are modified, for example by restriction digestion, prior to insertion into compatible vectors.
Since primers are synthesized in a 3′ to 5′ direction, failed or incomplete synthesis of these DNA oligos will have truncated 5′ sequences. Therefore, purification is recommended to remove not only excess reagents from the synthesis but also non–full-length DNA oligos, to ensure successful cloning of the desired PCR fragment.
In addition to insert preparation, PCR is a useful method after cloning to screen for whether colonies carry the desired insert. Primers can be designed to determine the insert’s presence as well as orientation in the vector (learn more about PCR colony screening).
One of the benefits of PCR cloning is the ability to introduce desired mutations into the gene of interest via cloning, for mutagenesis studies. In site-directed mutagenesis, PCR primers are designed to incorporate base substitutions, deletions, or insertions within a specific sequence. In the schemes shown in Figure 5, the primers are directed at a sequence that has already been cloned in a plasmid. The PCR product, containing the introduced mutation, is then self-ligated to regenerate a circular plasmid and used to transform competent cells (App note: Site-directed mutagenesis).
Figure 5. PCR in site-directed mutagenesis. Non-overlapping primers are employed in this approach (red asterisk = mutated nucleotide, grey line = deleted sequence, blue line = inserted sequence). Alternative primer designs such as those with 5′ overlapping sequences may also be considered [4-6].
Important considerations for site-directed mutagenesis experiments include:
- Primer design
- DNA polymerase choice
- PCR parameters
Primer design: In designing mutagenic primers, it is desirable to place the mutated sequence near the middle of the primer, or at least 7–8 nt away from the 3′ end. This allows efficient 3′ extension and prevents mismatch base repair (3′→5′ exonuclease activity) by the DNA polymerase. Purification of PCR primers is recommended to maximize mutagenesis and cloning efficiency (learn more: Oligo technical resources).
DNA polymerase choice: The use of a high-fidelity DNA polymerase is critical for generating PCR fragments with the desired mutation without introducing unintended errors. Also, the chosen DNA polymerase must be able to amplify the entire length of the template DNA (e.g., to obtain a full-length mutated plasmid).
PCR parameters: PCR error rates are lower with shorter PCR targets and fewer PCR cycles. To preserve sequence accuracy when amplifying longer DNA and/or to obtain high yields from fewer PCR cycles, an extremely high-fidelity DNA polymerase with high processivity is recommended (learn more about DNA polymerase characteristics).
To introduce multiple mutation sites, mutagenic primers with overlapping homologous sequences can be designed for PCR. The homologous ends of the amplicons then recombine directionally, resulting in a plasmid with desired multiple mutations (Figure 6). This approach can be followed not only to generate mutations in a plasmid that is too long to amplify by a single PCR, but also to avoid the higher error rates of long PCR amplification (learn more about long PCR).
Figure 6. Site-directed mutagenesis using PCR primers with mutant sequences and homologous end sequences. The approach shown here depicts the mechanism of the Invitrogen GeneArt Site-Directed Mutagenesis Kits, where the square blocks represent recombination and mutagenic sites.
PCR can be employed to investigate locus-specific methylation. In a method called methylation-specific PCR (MSP), two primer pairs are designed to differentiate the methylation state of the locus of interest [7,8].
In MSP, DNA samples are first treated with bisulfite to convert unmethylated cytosine (C) to uracil (U). Methylated cytosine (m5C) remains unaffected by the bisulfite treatment. To detect the methylated sites, one pair of primers is designed with guanine (G) to pair with m5C in the target sequence; to detect the unmethylated sites, another pair of primers is designed with adenine (A) to pair with U in the bisulfite-converted molecules (and then pair with thymine (T) in subsequent PCR cycles). Positive PCR amplification resulting from primer binding is used to determine the methylation state of the locus (Figure 7). (Also learn about methylation analysis by restriction enzymes.)
Figure 7. Methylation-specific PCR. In the first step, DNA samples are treated with bisulfite to convert unmethylated cytosine to uracil. Two sets of PCR primers (methylation and non-methylation) are designed to differentiate the methylation state of a locus based on amplification of the bisulfite-treated DNA. Unmethylated DNA is present as single strands after bisulfite treatment due to G-U mismatches, and only one strand of DNA is shown here for simplification.
Since MSP relies heavily on the primer’s specificity toward the bisulfite-converted sequence, primer design plays a critical role in experimental success. First, primer-binding sites must contain methylation-susceptible residues so that methylated vs. unmethylated sequences can be detected. Second, the non-methylation primers are typically AT-rich and thus need to be long (e.g., >30 nt) with Tm ≥60°C to enable specific annealing. In addition, high-AT sequences often favor primer-dimer formation, mismatch hybridization, DNA polymerase slippage, and amplification bias. Therefore, the selected DNA polymerase must be able to amplify templates with a wide range of AT/GC content. Third, primer specificity must be empirically tested with control DNAs of known and unknown methylation states to assess false positive results. To help discriminate methylation states by base-pair mismatches, it is advisable to design methylation and non-methylation primers with a pair of G-A or T-C at their 3′ ends (Figure 7).
In addition to amplifying AT-rich sequences, a DNA polymerase must be able to read through U residues in the DNA after bisulfite treatment. Most high-fidelity DNA polymerases are not suitable for MSP (unless specially modified) due to the presence of a uracil-binding pocket from their Archaean origins. Similarly, the presence of U in the template sequence does not allow UDG treatment for prevention of PCR carryover contamination.
Instead of endpoint PCR, real-time PCR can be used with MSP for more quantitative analysis of methylation. With real-time PCR, melting curve analysis of PCR amplicons is an alternative PCR-based approach for detecting the methylation state of the locus of interest.
PCR is a relatively simple approach for enriching template DNA for sequencing. High-fidelity PCR is highly recommended for preparation of sequencing templates, in order to maintain DNA sequence accuracy.
In Sanger sequencing, PCR-amplified fragments are purified and subjected to the sequencing reactions. The PCR primers can be tagged at their 5′ ends with commonly used binding site for sequencing primers (e.g., M13 or T7 “universal primer” binding site) to simplify the sequencing workflow (Figure 8).
Figure 8. Preparation of PCR amplicons for Sanger sequencing. PCR primers can be tagged with common sequencing primer sites to facilitate the workflow.
In next-generation sequencing (NGS), PCR is widely used to construct DNA sequencing libraries. In NGS library preparation, DNA samples are enriched by PCR (when the starting quantity is limited) and tagged with sequencing adaptors (along with unique barcodes or indices for multiplexing) (Figure 9). In addition to high fidelity, the DNA polymerase(s) employed should display minimum bias in amplification to provide representative coverage for the sequencing libraries.
Figure 9. PCR in preparing DNA samples for next-generation sequencing.
Medical, forensic, and applied sciences
In addition to basic research, PCR-based technologies are used every day in clinical diagnostics, forensic investigations, and agricultural biotechnology. These applications require reliable performance, superb sensitivity, and stringent specifications. As such, thermal cyclers and reagents must be compliant to and specially designed for these purposes. Examples of molecular diagnostics include genetic testing, detection of oncogenic mutations, and testing for infectious diseases. In forensics, human identification by PCR relies on amplification of unique short tandem repeats (STRs) on gDNA to differentiate individuals. In agriculture, PCR plays an integral role in food pathogen detection, plant genotyping for breeding, and GMO testing.
In conclusion, since its introduction in the 1980s, PCR continues to prove to be a useful tool with broad applications in discovery biology, medical diagnostics, forensics, and agriculture.
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