Thursday, May 07, 2015

Lab Series# 5: DNA amplification in Lab

Every individual personnel, involved with biology as at least heard of a PCR. Though originally argued to be the brain child of K Mullis (For which he was awarded Nobel Prize), Dr Khorana at least deserves credit. And what better topic to begin blogging about Clinical Lab Science, than talk about PCR technique.

PCR or polymerase chain reaction is a method for amplifying specific DNA sequences. Standard PCR involves amplification of a single DNA sequence that is less than 5 kb in length and is useful for a variety of applications, such as cycle sequencing, cloning, mutation detection etc. Long PCR is used for the amplification of a single sequence that is longer than 5 kb and up to 40 kb in length. A PCR cycle consists of three steps.
  1. Melting of DNA
  2. Hybridization of primers
  3. DNA synthesis or extension of primers
Melting of DNA:

Fig 1: Denaturation of DNA.
Source
Also known as strand separation or denaturation. The two strands of the parent DNA molecule are separated by heating the solution to 92-95º C. The temperature required for the melting of DNA can be approximately calculated by using the formula; TmC = 2(A/T) + 4(G/C). Melting temperature, or Tm, is defined for a given DNA duplex as the temperature at which half of the strands are hybridized and half of the strands are not hybridized. The Tm of a given primer/template combination depends on primer concentration, template concentration, and salt concentration. Generally, the template concentration is considered to be negligible compared with the primer concentration, so the formula for calculating Tm is simplified such that it does not contain a parameter for template concentration. Tm can be expressed as

Tm= {ΔH / (ΔS+ Rln(c)}-{273.15 + 16.6 log [Na+]}

The primer concentration is c; the ΔH and ΔS refer to the total enthalpy and entropy of hybridization, respectively; and [Na+] is the sodium ion concentration but can refer to the total concentration of most monovalent cations (such as K+). If there is Mg2+ or other divalent cations such as Mn2+, the conversion is generally accepted as Na+ = 4 × [Mg2+] 2

Hybridization of primers:

Fig 2: Hybridization of primers.
Source
The step is also known as Annealing of primers. The solution is cooled to 54-65º C to allow each primer to bind to a DNA strand. One primer hybridizes to the 3’end of the target on one strand, and the other primer hybridizes to the 3’ end on the complementary target strand. Two primers- Forward primer and the Reverse primers are used.

DNA synthesis or extension of primers:

The solution is heated to 72-75º C, the optimal temperature for Taq DNA polymerase. The polymerase elongates both primers in the direction of the target sequence because DNA synthesis is in the 5’-to-3’ direction. For obvious reasons the desired site is replicated in exact lengths from 3rd Cycle only. For a explanation check this link. These three steps constitute one cycle of the PCR amplification. The duplexes are heated to begin the second cycle, which produces four duplexes, and then the third cycle is initiated. The thermophilic DNA polymerases, like other DNA polymerases, catalyze template directed synthesis of DNA from nucleotide triphosphates. A primer having a free 3' hydroxyl is required to initiate synthesis and magnesium ion is necessary. In general, they have maximal catalytic activity at 75 to 80 C, and substantially reduced activities at lower temperatures. At 37C, Taq polymerase has only about 10% of its maximal activity.

The advantages of using Taq Polymerases include
  • Not permanently destroyed at 94ºC
  • Optimal temperature is 72ºC
  • The disadvantages include
The disadvantages include
  • Does not have proof reading ability
  • Error rate of nearly 1 in 2 X 104 bases
A basic PCR run can be broken up into three phases

Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.

Fig 3: Phases in PCR reaction. Source
Linear (High Variability): The reaction components are being consumed, the reaction is slowing, and products are starting to degrade.

Plateau (End-Point: Gel detection for traditional methods): The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.

From the very first methods of PCR, we have now come a long way to more complicated PCR. There are a large varieties of PCR techniques available in the current market. There are several variations of PCR technique. One of the most commonly used is Real time PCR (qPCR), especially in diagnostics.

Real time PCR, as the name suggests, indicates results are available to the experimenter in real time. In conventional PCR, the end products are analysed (for example by running on a agarose gel). In contrast, qPCR detects the amplified DNA product as the amplification is on the process, providing results in Real time. The qPCR principle is of 2 basic types based on the chemistry involved in the reaction

1. Fluorescent Primer and Probe-Based
2. Dye based

There are different varieties of probe based chemistry available from various kits. The most commonly used is the TaqMan method and Molecular beacon method. Other methods that are available in market includes Molecular beacons, Hybridization probes., Eclipse probes, Scorpion probes, LUX primer, BD QZyme primers etc. Irrespective of the type of probe and primer that is used, they work on a common principle of FRET (fluorescence resonance energy transfer) or other fluorescence- quenching mechanisms, to ensure that only specific fluorescence related to amplified product is detected. Additionally, in contrast to dye based chemistry, the probe/ primer based qPCR are more specific allowing multiplex operations to performed.

For the purpose of discussion and understanding, I will elaborate only on TaqMan assay. For those interested in other probes, please refer to links provided in further reading.

Fig 4: TaqMan Probe. Source
The TaqMan probe is a sequence specific, fluorescent labelled oligonucleotide sequence. A reporter fluorescent dye (in the Fig 1 represented as R) is lined to 5' end of probe and a Quencher (in the Fig 1 represented as Q) at 3' is used. The sequence of the probe is not more than 10-12 bp and is specific for the target DNA sequence. In presence of suitable excitation wavelength the reporter fluoresce but is quenched immediately by FRET. This leads to lack of signals. At the annealing / extension step of the amplification reaction, the probe binds to specific sequence lying in between the target DNA sequence. The reaction proceeds, and nucleotides are added. When the reaction meets the probe the probe sequence is cleaved and there is separation of R and Q. The quencher is no more able to quench the signals generated and is detected by using suitable detectors. Since this chemistry uses DNA polymerase that can attack the 5' R through nuclease activity (dsDNA-specific 5'—>3' exonuclease activity), the technique is also known as 5'- nuclease method.

Reporter
Quencher
FAM (Carboxy fluoerescin)
Black Hole Quencher-1(BHQ-1)
TET
BHQ1, DABCYL (N-[4-(4-dimethylamino)phenylazo] benzoic acid)
Texas Red
BHQ2, BHQ3, DABCYL
ROX
BHQ2, BHQ3, DABCYL

Fig 5: Principle of qPCR using TaqMan Probe. Source
Let me talk about one more of technique of great interest- Eclipse probe. The probe is similar to TaqMan with a quencher and Reporter but has an additional site called as MGB (Minor groove binder). In unbound conditions probe adopts a conformation that brings the reporter and quencher together, quenching the reporter. During amplification, probe hybridizes to the target with the help of the minor groove binder. This provides additional specificity. This leads to changes in the probe alignment and converts to linearized form, separating the reporter and quencher and allowing the reporter to fluoresce. This is detected as a emission signal.

Fig 6: Eclipse probe. Source
Dye based chemistry is a much more older method. The method uses a fluoerescing chemical that can bind to DNA (Inter-calating agent). As the DNA multiplies, there is more dsDNA. This can be detected by using the dye which binds dsDNA. The method carries the advantage of not having to design a specific probe, but comes at cost of specificity (Binds any dsDNA) and mulitplexing is not an option here. Almost universally, the preferred dye is SYBR Green I. SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain. The resulting DNA-dye-complex absorbs blue light and emits green light. But its not the only dye that is available. Dyes such as SYTO9, SYTO-82 and SYTO-13 have similar properties as that of SYBR. All these dyes do not inhibit PCR, but show a preferential binding to GC-rich regions and influence melting temperature. This has important applications in melt curve analysis.

Fig 7: SYBR Green I
Source
More recently a large variety of dyes have been developed that has outperformed the SYBR class of dye. Of all, a dye- EvaGreen has shown great promise. Multiple studies show it has higher stability and better compliance than that of SYBR class of dyes. Moreover, the absorption and emission spectra of EvaGreen are very much similar to that of SYBR Green I.

PCR is just one of the methods to amplify DNA. The drawback of this method is the requirement of specialized equipment. This reduces its applicability in the field. Over the years isolathermal methods have slowly gained popularity in DNA amplification especially for field work settings. As an example of it, let us look at LAMP assay.

Fig 8: Some commonly used techniques in nucleic acid amplification.
Loop mediated isothermal amplification (LAMP) is an alternative for PCR, used to amplify the DNA. In contrast to PCR, this doesn't require a thermal cycler. The process of genome amplification is done at a uniform temperature. Hence the terminology- isothermal amplification. During the reaction a loop based structure is produced, hence referred as Loop mediated. The method was first developed by Eiken Chemical Co., Ltd in 1998 and first peer reviewed publication was described by Tsugunori Notomi and group. The original method utilizes a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.

The LAMP uses 4 primers- Two forward primers: Forward Inner Primer (FIP), Forward Outer Primer (FOP) and 2 backward primers: Backward Inner Primer (BIP), Backward Outer Primer (BOP).

The first step of amplification is to make single stranded DNA from double stranded DNA. This is accomplished in PCR by heating. The same is achieved in LAMP by using Bst polymerase which can displace the dsDNA efficiently at temperatures less than 65 C. Bst is derived from Bacillus stearothermophilus and contains the 5´ → 3´ polymerase activity. Its activity diminishes at temperatures higher than 65 C. Bst 2.0 DNA Polymerase is an in silico designed homologue of Bacillus stearothermophilus DNA Polymerase I, with improved properties in comparison to the earlier type. Both types are tolerant various inhibitors usually associated with diagnostic specimens. This serves as an advantage over the PCR Taq polymerases.

The LAMP reaction is started by adding the Bst polymerase, which opens the DNA and allows primer to be hybridized. Further the polymerase, simultaneously synthesizes the other DNA strand. In the process a stem loop is formed which automatically primes the next round of reaction. A fantastic step by step explanation of how exactly this happens, is given here. Since all steps are combined into a single step which automatically can cycle by itself, the need to heat cycling the reaction is avoided. The technique comes with an added advantage. By virtue of multiple primer being used, the reaction is more specific with better product yield in comparison to PCR.

To state in nutshell, the reaction takes place in a single tube containing appropriate buffer, extracted target DNA, Bst polymerase and 4 primers. The tube is incubated at 64°C. The reaction can be detected easily by addition of DNA intercalating agent such as SYBR green.

Photo 1: Visual detection of LAMP product under UV light.
From left to right, tubes 1, 2, 7, and 8 are negative
and tubes 3, 4, 5, and 6 are positive. Source
In current technology, the detection is improvised by using a calcein system. It is known that the DNA replication process yields pyrophosphate ions as a by-product from the substrate dNTPs. The calcein in the reaction mixture initially combines with manganous ion (Mn2+) so as to remain quenched. As the amplification proceeds, manganous ion is deprived of calcein by the generated pyrophosphate ion (P2O74-resulting in fluorescence. The free calcein is can now combine with magnesium ions in the reaction mixture resulting in stronger emission.

The LAMP testing method can also be used for detection of RNA, by using a additional step of reverse transcription. The obvious advantages are that its a isothermal procedure and doesn' require any additional consumables or equipments. LAMP method has already gained popularity in detection of pathogens. It is of considerable importance in TB field testing.

I want to end with an additional note here. Other than the the common isothermal methods that I mentioned earlier, there are a few more that am aware of but hasn't gained much popularity. That includes- Nicking enzyme amplification reaction (NEAR), Helicase- dependent amplification (HDA), Recombinase polymerase amplification (RPA) etc.

Further Reading:

1. Kutyavin IV. Use of base modifications in primers and amplicons to improve nucleic acids detection in the real-time snake polymerase chain reaction. Assay Drug Dev Technol. 2011 Feb;9(1):58-68. Link

2. Thornton B, Basu C. Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ. 2011 Mar-Apr;39(2):145-54. Link

3. Eugeny A. Lukhtanov, Sergey G. Lokhov, Vladimir V. Gorn, Mikhail A. Podyminogin, and Walt Mahoney. Novel DNA probes with low background and high hybridization-triggered fluorescence. Nucleic Acids Res. 2007 March; 35(5): e30. Link

4. Anne C Eischeid. SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR. BMC Research Notes 2011, 4:263. Link

5. Tsugunori Notomi. Loop-mediated isothermal amplification of DNA. Nucl. Acids Res. (2000) 28 (12): e63. Link

6. Parida M, Sannarangaiah S, Dash PK, Rao PV, Morita K.Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol. 2008 Nov-Dec;18(6):407-21. Link

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