COLD-PCR

Last updated January 08, 2023

COLD-PCR (co-amplification at lower denaturation temperature PCR) is a modified polymerase chain reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA. The ability to preferentially amplify and identify minority alleles and low-level somatic DNA mutations in the presence of excess wildtype alleles is useful for the detection of mutations. Detection of mutations is important in the case of early cancer detection from tissue biopsies and body fluids such as blood plasma or serum, assessment of residual disease after surgery or chemotherapy, disease staging and molecular profiling for prognosis or tailoring therapy to individual patients, and monitoring of therapy outcome and cancer remission or relapse. Common PCR will amplify both the major (wildtype) and minor (mutant) alleles with the same efficiency, occluding the ability to easily detect the presence of low-level mutations. The capacity to detect a mutation in a mixture of variant/wildtype DNA is valuable because this mixture of variant DNAs can occur when provided with a heterogeneous sample – as is often the case with cancer biopsies. Currently, traditional PCR is used in tandem with a number of different downstream assays for genotyping or the detection of somatic mutations. These can include the use of amplified DNA for RFLP analysis, MALDI-TOF (matrix-assisted laser-desorption–time-of-flight) genotyping, or direct sequencing for detection of mutations by Sanger sequencing or pyrosequencing. Replacing traditional PCR with COLD-PCR for these downstream assays will increase the reliability in detecting mutations from mixed samples, including tumors and body fluids.

Method overviews

Conventional PCR compared to COLD-PCR. COLD-PCR.jpg — Conventional PCR compared to COLD-PCR.

The underlying principle of COLD-PCR is that single nucleotide mismatches will slightly alter the melting temperature (Tm) of the double-stranded DNA. Depending on the sequence context and position of the mismatch, Tm changes of 0.2–1.5 °C (0.36–2.7 °F) are common for sequences up to 200bp or higher. Knowing this the authors of the protocol took advantage of two observations:

Each double-stranded DNA has a 'critical temperature' (Tc) lower than its Tm. The PCR amplification efficiency drops measurably below the Tc.
The Tc is dependent on DNA sequence. Two template DNA fragments differing by only one or two nucleotide mismatches will have different amplification efficiencies if the denaturation step of PCR is set to the Tc.

Keeping these principles in mind the authors developed the following general protocol:

Denaturation stage. DNA is denatured at a high temperature –usually 94 °C (201 °F).
Intermediate annealing stage. Set an intermediate annealing temperature that allows hybridization of mutant and wildtype allele DNA to one another. Because the mutant allele DNA forms the minority of DNA in the mixture they will be more likely to form mismatch heteroduplex DNA with the wildtype DNA.
Melting stage. These heteroduplexes will more readily melt at lower temperatures. Hence they are selectively denatured at the Tc.
Primer annealing stage. The homo-duplex DNA will preferentially remain double stranded and not be available for primer annealing.
Extension stage. The DNA polymerase will extend complementary to the template DNA. Since the heteroduplex DNA is used as template, a larger proportion of minor variant DNA will be amplified and be available for subsequent rounds of PCR.

There are two forms of COLD-PCR that have been developed to date. Full COLD-PCR and fast COLD-PCR.

Full

Full COLD-PCR is identical to the protocol outlined above. These five stages are used for each round of amplification.

Fast

Fast COLD-PCR differs from Full COLD-PCR in that the denaturation and intermediate annealing stages are skipped. This is because, in some cases, the preferential amplification of the mutant DNA is so great that ensuring the formation of the mutant/wildtype heteroduplex DNA is not needed. Thus the denaturation can occur at the Tc, proceed to primer annealing, and then polymerase-mediated extension. Each round of amplification will include these three stages in that order. By utilizing the lower denaturation temperature, the reaction will discriminate toward the products with the lower Tm – i.e. the variant alleles. Fast COLD-PCR produces much faster results due to the shortened protocol, while Full COLD-PCR is essential for amplification of all possible mutations in the starting mixture of DNA.

Two-round COLD-PCR is a modified version of Fast COLD-PCR. During the second round of Fast COLD-PCR nested primers are used. This improves the sensitivity of mutation detection compared to one-round Fast COLD-PCR.^[1]

Uses

COLD-PCR has been used to improve the reliability of a number of different assays that traditionally use conventional PCR.

RFLP

A restriction fragment length polymorphism results in the cleavage (or absence thereof) of DNA for a specific mutation by a selected restriction enzyme that will not cleave the wildtype DNA. In a study using a mixture of wildtype and mutation containing DNA amplified by regular PCR or COLD-PCR, COLD-PCR preceding RFLP analysis was shown to improve the mutation detection by 10-20 fold.^[2]

Sanger sequencing

Sanger sequencing recently was used to evaluate the enrichment of mutant DNA from a mixture of 1:20 mutant:wildtype DNA. The variant DNA containing a mutation was obtained from a breast cancer cell line known to contain p53 mutations.^[1] Comparison of Sanger sequencing chromatograms indicated that the mutant allele was enriched 13 fold when COLD-PCR was used compared to traditional PCR alone.^[1] This was determined by the size of the peaks on the chromatogram at the variant allele location.

As well, COLD-PCR was used to detect p53 mutations from lung-adenocarcinoma samples. The study was able to detect 8 low level (under 20% abundance) mutations that would likely have been missed using conventional methods that don't enrich for variant sequence DNA.

Pyrosequencing

Similar to its use in direct Sanger sequencing, with pyrosequencing COLD-PCR was shown to be capable of detecting mutations that had a prevalence 0.5–1% from the samples used.^[3] COLD-PCR was used to detect p53 and KRAS mutations by pyrosequencing, and was shown to outperform conventional PCR in both cases.

MALDI-TOF

The same research group that developed COLD-PCR and used it to compare the sensitivity of regular PCR for genotyping with direct Sanger sequencing, RFLP, and pyrosequencing, also ran a similar study using MALDI-TOF as a downstream application for detecting mutations. Their results indicated that COLD-PCR could enrich mutation sequences from a mixture of DNA by 10–100 fold and that mutations with an initial prevalence of 0.1–0.5% would be detectable.^[4] Compared to the 5–10% low-level detection rate expected with traditional PCR.

QPCR

COLD-PCR run on a quantitative PCR machine, using TaqMan probes specific for a mutation, was shown to increase the measured difference between mutant and wildtype samples.^[4]

Advantages

Single-step method capable of enriching both known and unknown minority alleles irrespective of mutation type and position
Does not require extra costly reagents or specialized machinery
Better than conventional PCR for the detection of mutations in a mixed sample
Does not significantly increase experiment run time compared to conventional PCR

Disadvantages

Optimal Tc must be measured and determined for each amplicon, adding an extra step to conventional PCR-based procedures
Requirement for precise denaturation temperature control during PCR to within ± 0.3 °C (0.54 °F)
A suitable critical temperature may not be available that differentiates between mutant and wildtype DNA sequences
Restricted to analyzing sequences smaller than approximately 200bp
Vulnerable to polymerase-introduced errors
Variable overall mutation enrichment dependent on DNA position and nucleotide substitution
No guarantee that all low-level mutations will be preferentially enriched

History

COLD-PCR was originally described by Li et al. in a Nature Medicine paper published in 2008 from Mike Makrigiorgos's lab group at the Dana Farber Cancer Institute of Harvard Medical School.^[2] As summarized above, the technology has been used in a number of proof-of-principle experiments and medical research diagnostic experiments.

Recently, the COLD-PCR technology has been licensed by Transgenomic, Inc. The licensing terms include the exclusive rights to commercialize the technology combined with Sanger sequencing. The plans are to develop commercial applications that will allow for rapid high-sensitivity detection of low-level somatic and mitochondrial DNA mutations.^[5]

Alternatives

Other technologies are available for the detection of minority DNA mutations, and these methods can be segregated into their ability to enrich for and detect either known or unknown mutations.^[6]

Related Research Articles

The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

<span class="mw-page-title-main">Sanger sequencing</span> Method of DNA sequencing developed in 1977

Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method for approximately 40 years. It was first commercialized by Applied Biosystems in 1986. More recently, higher volume Sanger sequencing has been replaced by next generation sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use for smaller-scale projects and for validation of deep sequencing results. It still has the advantage over short-read sequencing technologies in that it can produce DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%. Sanger sequencing is still actively being used in efforts for public health initiatives such as sequencing the spike protein from SARS-CoV-2 as well as for the surveillance of norovirus outbreaks through the Center for Disease Control and Prevention's (CDC) CaliciNet surveillance network.

Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual.

Heteroduplex analysis (HDA) is a method in biochemistry used to detect point mutations in DNA since 1992. Heteroduplexes are dsDNA molecules that have one or more mismatched pairs, on the other hand homoduplexes are dsDNA which are perfectly paired. This method of analysis depend up on the fact that heteroduplexes shows reduced mobility relative to the homoduplex DNA. heteroduplexes are formed between different DNA alleles. In a mixture of wild-type and mutant amplified DNA, heteroduplexes are formed in mutant alleles and homoduplexes are formed in wild-type alleles. There are two types of heteroduplexes based on type and extent of mutation in the DNA. Small deletions or insertion create bulge-type heteroduplexes which is stable and is verified by electron microscope. Single base substitutions creates more unstable heteroduplexes called bubble-type heteroduplexes, because of low stability it is difficult to visualize in electron microscopy. HDA is widely used for rapid screening of mutation of the 3 bp p.F508del deletion in the CFTR gene.

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.

An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay. It is a common tool used in genetic testing, forensics, and Molecular Biology research.

Digital polymerase chain reaction is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. A "digital" measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences — such as copy number variants and point mutations — and it is routinely used for clonal amplification of samples for next-generation sequencing.

A nucleic acid test (NAT) is a technique used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissue, urine, etc. NATs differ from other tests in that they detect genetic materials rather than antigens or antibodies. Detection of genetic materials allows an early diagnosis of a disease because the detection of antigens and/or antibodies requires time for them to start appearing in the bloodstream. Since the amount of a certain genetic material is usually very small, many NATs include a step that amplifies the genetic material—that is, makes many copies of it. Such NATs are called nucleic acid amplification tests (NAATs). There are several ways of amplification, including polymerase chain reaction (PCR), strand displacement assay (SDA), or transcription mediated assay (TMA).

Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. The temperature at which 50% of DNA is denatured is known as the melting temperature. Measurement of melting temperature can help us predict species by just studying the melting temperature. This is because every organism has a specific melting curve.

The versatility of polymerase chain reaction (PCR) has led to a large number of variants of PCR.

High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples. It was discovered and developed by Idaho Technology and the University of Utah. It has advantages over other genotyping technologies, namely:

Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.

Multiple Annealing and Looping Based Amplification Cycles (MALBAC) is a quasilinear whole genome amplification method. Unlike conventional DNA amplification methods that are non-linear or exponential, MALBAC utilizes special primers that allow amplicons to have complementary ends and therefore to loop, preventing DNA from being copied exponentially. This results in amplification of only the original genomic DNA and therefore reduces amplification bias. MALBAC is “used to create overlapped shotgun amplicons covering most of the genome”. For next generation sequencing, MALBAC is followed by regular PCR which is used to further amplify amplicons.

Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. In medicine the technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients, and in agricultural biosecurity similarly to monitor crop- and livestock disease, estimate risk, and decide what quarantine measures must be taken.

SNV calling from NGS data is any of a range of methods for identifying the existence of single nucleotide variants (SNVs) from the results of next generation sequencing (NGS) experiments. These are computational techniques, and are in contrast to special experimental methods based on known population-wide single nucleotide polymorphisms. Due to the increasing abundance of NGS data, these techniques are becoming increasingly popular for performing SNP genotyping, with a wide variety of algorithms designed for specific experimental designs and applications. In addition to the usual application domain of SNP genotyping, these techniques have been successfully adapted to identify rare SNPs within a population, as well as detecting somatic SNVs within an individual using multiple tissue samples.

Surveyor nuclease assay is an enzyme mismatch cleavage assay used to detect single base mismatches or small insertions or deletions (indels).

Duplex sequencing is a library preparation and analysis method for next-generation sequencing (NGS) platforms that employs random tagging of double-stranded DNA to detect mutations with higher accuracy and lower error rates.

References

1 2 3 Li J, Milbury CA, Li C, Makrigiorgos GM (November 2009). "Two-round COLD-PCR-based Sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma". Human Mutation. 30 (11): 1583–90. doi:10.1002/humu.21112. PMC 2784016 . PMID 19760750.
1 2 Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (May 2008). "Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing". Nature Medicine. 14 (5): 579–84. doi:10.1038/nm1708. PMID 18408729. S2CID 205385306.
↑ Zuo Z, Chen SS, Chandra PK, et al. (August 2009). "Application of COLD-PCR for improved detection of KRAS mutations in clinical samples". Modern Pathology. 22 (8): 1023–31. doi: 10.1038/modpathol.2009.59 . PMID 19430420.
1 2 Li J, Makrigiorgos GM (April 2009). "COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing". Biochemical Society Transactions. 37 (2): 427–32. doi:10.1042/BST0370427. PMID 19290875.
↑ Laboratorytalk editorial team (Oct 2009). "Transgenomics licenses COLD-PCR technology". Archived from the original on 2011-07-18. Retrieved 2010-03-04.
↑ Milbury CA, Li J, Makrigiorgos GM (April 2009). "PCR-Based Methods for the Enrichment of Minority Alleles and Mutations". Clinical Chemistry. 55 (4): 632–40. doi:10.1373/clinchem.2008.113035. PMC 2811432 . PMID 19201784.

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[nested-1] 1 2 3 Li J, Milbury CA, Li C, Makrigiorgos GM (November 2009). "Two-round COLD-PCR-based Sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma". Human Mutation. 30 (11): 1583–90. doi:10.1002/humu.21112. PMC 2784016 . PMID 19760750.

[original-2] 1 2 Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (May 2008). "Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing". Nature Medicine. 14 (5): 579–84. doi:10.1038/nm1708. PMID 18408729. S2CID 205385306.

[3] Zuo Z, Chen SS, Chandra PK, et al. (August 2009). "Application of COLD-PCR for improved detection of KRAS mutations in clinical samples". Modern Pathology. 22 (8): 1023–31. doi: 10.1038/modpathol.2009.59 . PMID 19430420.

[review-4] 1 2 Li J, Makrigiorgos GM (April 2009). "COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing". Biochemical Society Transactions. 37 (2): 427–32. doi:10.1042/BST0370427. PMID 19290875.

[5] Laboratorytalk editorial team (Oct 2009). "Transgenomics licenses COLD-PCR technology". Archived from the original on 2011-07-18. Retrieved 2010-03-04.

[6] Milbury CA, Li J, Makrigiorgos GM (April 2009). "PCR-Based Methods for the Enrichment of Minority Alleles and Mutations". Clinical Chemistry. 55 (4): 632–40. doi:10.1373/clinchem.2008.113035. PMC 2811432 . PMID 19201784.

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v t e Polymerase chain reaction techniques
Procedure	Initialization Cycle Denaturation Annealing Elongation Final elongation Final hold
Polymerase	Klenow T4 and T7 Taq Tth Pfu Vent Pwo Tli Tfu
Optimization ＆ variants	Quantitative polymerase chain reaction (qPCR) Reverse transcription polymerase chain reaction (RT-PCR) Inverse polymerase chain reaction (inverse PCR) Nested polymerase chain reaction (nested PCR) Touchdown polymerase chain reaction Hot start PCR Overlap extension polymerase chain reaction (OE-PCR) Multiplex polymerase chain reaction (multiplex PCR) Multiplex ligation-dependent probe amplification (MLPA) Co-amplification at lower denaturation temperature PCR (COLD-PCR) Random amplification of polymorphic DNA (RAPD)
History ＆ people	Kary Mullis Kjell Kleppe Alice Chien