Nucleic acid amplification: Alternative methods of polymerase chain reaction.

Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

Nucleic acid amplification is a pivotal process in biotechnology and molecular biology and has been widely used in research, medicine, agriculture and forensics. Polymerase chain reaction (PCR) was the first nucleic acid amplification method developed and until now has been the method of choice since its invention by Mullis.[1] PCR is the preferred method for application oriented fields involving nucleic acid amplification for its simplicity, easier methodology, extensively validated standard operating procedure and availability of reagents and equipments. However, PCR has a good no of limitations, including high cost of equipment, contamination chances, sensitivity to certain classes of contaminants and inhibitors, requirement of thermal cycling etc.[2] These limitations gave birth to alternative methods such as loop mediated isothermal amplification (LAMP), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA) etc., most of which are isothermal nucleic acid amplification methods obviating the need of thermal cycler. These methods offer potential advantages over PCR for speed, cost, scale or portability. In this review, most potential alternative methods of PCR are discussed in terms of their principles and applications.

Alternative Methods of Polymerase Chain Reaction

Several alternative amplification methods have been developed already, such as LAMP,[3] 3SR[4] or NASBA,[5] strand displacement amplification (SDA)[6] and RCA,[7] ligase chain reaction (LCR).[8]

Alternative methods are described here in brief.

Loop Mediated Isothermal Amplification

LAMP is a specific, simple, rapid and cost-effective isothermal nucleic acid amplification method. LAMP has an improved simple visual amplicon detection system. LAMP relies on the auto-cycling strand displacement deoxyribonucleic acid (DNA) synthesis which is carried out at 60-65°C for 45-60 min in the presence of Bacillus stearothermophylus (Bst) DNA polymerase, deoxyribonucleotide triphosphate (dNTPs), specific primers and the target DNA template. The LAMP method employs a DNA polymerase with high strand displacement activity and a set of four specially constructed primers (two inner and two outer primer) that recognize six distinct sequences on the target DNA. The mechanism of the LAMP amplification reaction includes three steps: Production of starting material, cycling amplification and elongation and recycling [Figure 1].[3] High-level of precision can be attained without expensive equipments. There are fewer and simpler sample preparation steps compared with conventional PCR and real-time PCR. Substantial alteration of the fluorescence of the reaction tube can be visualized without costly specialized equipment as the signal recognition system is highly sensitive. LAMP is a one-step amplification reaction taking only 30-60 min. LAMP is more resistant to various inhibitory compounds present in clinical samples than PCR, so there is no need for extensive DNA purification.[9] By combination with reverse transcription (RT), LAMP can amplify ribonucleic acid (RNA) sequences with high efficiency. It is highly sensitive and able to detect DNA at as few as six copies in the reaction mixture.[3] LAMP has the potential to be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and cost-effective diagnosis of infectious diseases.[2] LAMP is as suitable for DNA sequencing as PCR, in terms of both Sanger sequencing and Pyrosequencing.[1011]

Figure 1

Schematic description of loop mediated isothermal amplification assay

Nucleic Acid Sequence Based Amplification

NASBA, also known as 3SR[4] and transcription mediated amplification,[12] is an isothermal transcription-based amplification system. NASBA specifically designed for the detection of RNA targets. In some NASBA systems, DNA can also be amplified. The complete amplification reaction is performed at the predefined temperature of 41°C. Throughout the amplification reaction, constant temperature is maintained allowing each step of the reaction to proceed as soon as amplification intermediate formed. The exponential kinetic of the NASBA process is attributed by multiple transcription of RNA copies from a given DNA product, is intrinsically more efficient than DNA-amplification methods limited to binary increases per cycle.[13] This amplification system uses a consortium of three enzymes (avian myeloblastosis virus reverse transcriptase, RNase H and T7 DNA dependent RNA polymerase) leading to main amplification product of single-stranded RNA [Figure 2].[14] NASBA RNA product can be sequenced directly with a dideoxy method using RT and a labeled oligonucleotide primer. The length of the target sequence to be amplified efficiently is limited to approx 100-250 nucleotides. High-level of precision can be acquired without expensive equipments. NASBA amplicon detection step has significantly improved, incorporation of the use of enzyme-linked gel assay, enzymatic bead-based detection and electrochemiluminescent (ECL) detection, molecular beacon technology and fluorescent correlation spectroscopy.[15] In clinical use and pathogen detection, NASBA pose theoretically higher analytical sensitivity than reverse transcription-polymerase chain reaction RT-PCR making it an established diagnostic tool.[16] It has potential for detection and differentiation of viable cells through specific and sensitive amplification of messenger RNA, even against the background of genomic DNA.[17]

Figure 2

Principles of nucleic acid sequence based amplification

Strand Displacement Amplification

SDA, first described in 1992,[6] is an isothermic amplification method, which utilizes four different primers of which a primer containing a restriction site (a recognition sequence for HincII exonuclease) is annealed to the DNA template. An exonuclease-deficient fragment of Eschericia coli DNA polymerase 1 (exo-Klenow) elongates the primers. Each SDA cycle consists of (1) primer binding to a displaced target fragment, (2) extension of the primer/target complex by exoklenow, (3) nicking of the resultant hemiphosphothioate HincII site, (4) dissociation of HincII from the nicked site and (5) extension of the nick and displacement of the downstream strand by exo-Klenow [Figure 3].[18] This method can be performed at high temperatures. In a single reaction, 109 copies of target DNA can be produced in less than an hour. As with the other target amplification technologies (PCR, LCR, 3SR), only semi-quantitation is possible by this method. A major limitation of SDA is its inability to efficiently amplify long target sequences.[6] SDA is the basis for some commercial detection tests such as BDProbeTec (Becton Dickinson, Franklin Lakes, NJ, USA) and has been evaluated recently for the identification of Mycobacterium tuberculosis directly from clinical specimens.[19] The efficiency and robustness of this technology still remains to be proven in the large clinical studies. A recent development has been reported for real-time sequence specific DNA target detection using the fluorogenic reporter probes.[20]

Figure 3

Target generation scheme for strand displacement amplification

Multiple displacement amplification

The MDA is an isothermal, strand-displacing method based on the use of the highly processive and strand-displacing DNA polymerase from bacteriophage Ø29, in conjunction with modified random primers to amplify the entire genome with high-fidelity.[2122] It has been developed to amplify all DNA in a sample from a very small amount of starting material.[23] MDA by Ø29 DNA polymerase involves incubating Ø29 DNA polymerase, dNTPs, random hexamers and denatured template DNA at 30°C for 16-18 h. The enzyme is inactivated at 65°C for 10 min and the product DNA can be used directly in downstream applications [Figure 4].[24] In contrast to PCR-based methods no repeated cycling is required, but a short initial denaturation followed by the amplification step of 6-18 h and a final inactivation of the enzyme is needed [Figure 4]. This method can also be used to generate capture probes for microarrays, to produce highly pure DNA or even to amplify stored DNA.[25] MDA could be the method of choice when limited amounts of sample are available. Sensitivity and yields are high: About 20-30 μg of DNA can be obtained from as few as 1-10 copies of human genomic DNA.[26] In addition, MDA can be carried out directly from biological samples including crude whole blood and tissue culture cells.[25] MDA method presents several characteristics that, in combination with specific hybridization to macroarray and/or microarray, may be suitable for multiplex qualitative detection and identification. The utility of MDA has not been fully assessed for use in applications such as sample archiving, forensics and single cell clinical diagnostics.[24]

Figure 4

Schematic representation of multiple displacement amplification mechanism

Rolling Circle Amplification

RCA is an isothermal nucleic acid amplification method.[7272829] RCA technology enables the amplification of the probe DNA sequences more than 109 fold both in solution and on the solid phase at a single temperature. It has the ability to readily detect down to a few target-specific circularized probes in a test sample. In RCA reaction, numerous rounds of isothermal enzymatic synthesis is involved. Ø29 DNA polymerase extends a circle-hybridized primer by continuously progressing around the circular DNA probe of several dozen nucleotides to replicate its sequence over and over again [Figure 5].[3031] A major advantage of RCA is that unlike PCR, this technology is resistant to contamination and unlike some other isothermal technologies, requires little or no assay optimization. The capacity of RCA to yield the surface-bound amplification products offers significant advantages to in situ or microarray hybridization assays. In linear RCA, the product of amplification remains tethered to the target molecule. RCA seems well-suited to cell-and tissue-based assays in conjunction with the isothermal nature of the RCA reaction and the capability to localize multiple markers simultaneously. RCA is also suitable in cases where it is critical to maintain morphological information. RCA amplification permits the localization of signals, thus representing single molecules with specific genetic traits[732] or biochemical features.[2728]

Figure 5

Scheme for multiply-primed rolling circle amplification

RCA reactions exhibit an excellent sequence specificity that is favorable for genotyping or mutation detection and allows to unambiguously identifying DNA markers on the excessive unrelated background.[33] As compared with PCR, the RCA based DNA diagnostics are capable of higher multiplexity and they are less prone to amplification errors, thus allowing contamination-resistant detection of target molecules in a variety of testing formats. RCA has the potential for a highly localized isothermal detection of the designated sites on essentially intact duplex DNA with even superior specificity.[34] The simplicity and efficiency of RCA technology, along with ease and accuracy of quantitation, makes it amenable for miniaturization and automation in the high-throughput analysis.[35]

LCR[3637] is a cyclic DNA template-dependent amplification reaction. The method of DNA amplification is similar to PCR; however, LCR amplifies the probe molecule rather than producing amplicon through polymerization of nucleotides. LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme to drive the reaction. LCR uses two complementary pairs of oligonucleotides that hybridize in close proximity on the target fragment [Figure 6]. Only when the oligonucleotides correctly hybridize to the target sequence, the remaining nick between the oligonucleotides is ligated by a DNA ligase and a fragment equating to the total sequence of both oligonucleotides is generated. Once the probes have been ligated, the ligation product can serve as a template for annealing and future ligation. Like PCR, LCR requires a thermal circler to drive the reaction and each cycle results in a doubling of the target nucleic acid molecule.[38] The detection of LCR products can be performed by electrophoresis or by an enzyme-linked immunosorbent assay (ELISA) like microplate procedure or real-time detection.[39]

Figure 6

Ligase chain reaction

LCR can have greater specificity than PCR.[364041] It can be used for multiplex reactions rendering it suitable for detection of products by microarrays.[42] Its limitations come from the specificity of the ligase reaction, which is restricted to the region of the ligation junction.

This technique has one disadvantage over the detection of food pathogen that it can detect DNA from dead organism. The potential problem of this technology in addition to the risk of contamination[43] is clearly the lack of conformation, since only primer sequences are amplified. However, LDR sensitivity is limited, especially when rare targets need to be detected in the presence of high wild-type DNA background.[44] With this technology, SNPs can easily be differentiated.[45] It has been exploited to detect pathogens such as Neisseria gonorrhoneae, Chlamydia trachomatis and M. tuberculosis in human clinical specimens as well as detecting point mutations in C. trachomatis plasmid DNA, human immunodeficiency virus cloned fragments and in human sickle cell clinical samples.[46]

Helicase dependant amplification

HDA is an isothermal nucleic acid amplification method using the replication fork mechanism. Basic principle of HDA is the unwinding activity of a DNA helicase in the presence of adenosine tri phosphate.[47] Helicase separates the two strands of a DNA duplex generating single-stranded templates for the purpose of in vitro amplification of a target nucleic acid[48] and the displaced DNA strands are coated by single-stranded binding proteins. Two-sequence specific primers anneal to the 3’- end of each single-stranded DNA (ssDNA) template and exonuclease-deficient DNA polymerases produce double-stranded DNA by extending the primers annealed to the target DNA. Exponential amplification can be achieved if this process repeats itself at a single temperature [Figure 7]. This process allows multiple cycles of replication to be performed at a single incubation temperature, completely eliminating the need for thermo cycling equipment.[49] The HDA amplicons can be detected using gel electrophoresis, real-time format and ELISA.[50] High speed (100 bp/s) and processivity (10 kb/binding) are the major advantages of HDA.[51] HDA offers several advantages over other isothermal DNA amplification methods. It has a simple reaction scheme and can be performed at a single temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.[12]

Figure 7

Helicase-dependent amplification process. Step 1: The helicase unwinds deoxyribonucleic acid (DNA) duplexes. Step 2: The primers anneal to the single-stranded DNA. Step 3: The primers extended by DNA polymerase; one duplex is amplified and converted to two duplexes. The double-stranded DNAs are separated by helicase and this chain reaction repeats itself

Ramification amplification method

RAM is a novel isothermal nucleic acid amplification method. This technique is termed as RAM because the amplification power is derived from primer extension, strand displacement and multiple ramification (branching) points.[52] This method uses a specially designed circular probe (C-probe) in which the 3’ and 5’ ends are brought together in juxtaposition by hybridization to a target. The two ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed DNA circle. In the presence of an excess of primers (forward and reverse primers), bacteriophage Ø29 DNA polymerase extends the bound forward primer along the C-probe and displaces the downstream strand, thus generating a multimeric ssDNA by continuously rolling over the closed circular DNA, analogous to the “rolling circle” replication of bacteriophages in vivo. The multimeric ssDNA generated then serves as a template where multiple reverse primers hybridize, extend and displace downstream DNA and generate a large ramified (branching) DNA complex. This ramification process continues until all ssDNAs become double-stranded, resulting in an exponential amplification that distinguishes itself from the previously described nonexponential RCA [Figure 8]. By using a unique bacteriophage DNA polymerase, Ø29 DNA polymerase, that has an intrinsic high processivity, it is possible to achieve significant amplification within 1 h at 35°C.[53] As RAM is an isothermal amplification method and large multimeric products are generated, cell morphological characteristics are preserved while amplification products are better localized in the cells, making this method ideal for in situ amplification.[54] The RAM assay offers several advantages over other amplification techniques: (1) the primers readily bind to ssDNAs displaced by the DNA polymerase, enabling the reaction to be performed under isothermal conditions, obviating the need for a thermocycler; (2) generic primers amplify all probes with equal efficiency, resulting in better multiplex capability than conventional PCR;[52] both ends of the probe can be ligated regardless of the nature of target (DNA or RNA), eliminating the need for RT for detecting RNA and creating a uniform assay format for both RNA and DNA detection;[54] and (4) ligation requires that both probe termini hybridize with perfect matching, permitting the detection of a single-nucleotide polymorphism. It can be readily used in clinical laboratories for the detection of genes and infectious agents in various areas, such as Hematology, Oncology, infectious disease, Pathology, forensics, blood banks and genetic disease. In addition, it has great potential for use in field tests and doctors’ offices due to its simple and isothermal amplification format.[55]

Figure 8

Schematic representation of ramification amplification of ligated circular probe

Comparison of nucleic acid amplification methods

Each alternative method has their unique distinguishable properties as well as they have some common property.[56] A brief comparison of different key properties of nucleic acid amplification methods is given in Table 1.

Table 1

Comparison of nucleic acid amplification methods

Conclusion

Each of the alternate isothermal methods has their own limitations and their mechanisms are relatively complex, they are easy to perform and offer better sensitivity. Incorporation of real-time detection methods such as ECL, molecular beacon made them more competent to be used widely. In the future, some of these methods will be as acceptable and applicable as PCR.