Multiple Touchdown PCR (MT-PCR): A New Application of PCR for Better Precision and Stability
What is MT-PCR?
Polymerase chain reaction, or PCR, has been in use for nearly 30 years and is in use in all kinds of biochemical and medical laboratories. However, the most overwhelming problem that arises in PCR is the amplification of non-target DNA which can cause all kinds of trouble, not the least being false positive results. Fortunately, PCR techniques have constantly improved over the years and a new PCR technique – Multiple Touchdown PCR (MT-PCR) has been developed. MT-PCR combines features of both multiple PCR and touchdown PCR. Several primers are used to test multiple DNA targets in one PCR experiment. The annealing temperature is reduced from an original set point by 0.5~1°C for each cycle until the best annealing temperature is reached.
MT-PCR, which was presented in a new study in 2016*(1), was developed for the detection of resistance genes. MT-PCR successfully identifies several different target bacterial DNAs, it also prevents the reaction from amplifying non-target DNA and consequent false positive results.
How does MT-PCR solve the issue of multiple PCR?
In a regular PCR experiment, the target is a single DNA template and a specific primer will extend the template in 20 reaction cycles. Much time can be saved by using multiple primers and target DNAs in one reaction where we can extend several target DNAs in a single PCR at the same time*(2).
However, the use of multiple PCR has a serious disadvantage, it is not easy to avoid cross-amplification of non-target DNA in a mixture of primers and DNAs. Therefore we have to select the specific target DNAs in advance before we do the multiple PCR. Only target DNAs with different length of DNA pairs are selected for multiple PCR.
Although multiple PCR saves time, it still cannot prevent errors caused by the cross amplification of non-target DNA. That is why we need MT-PCR. The big difference between MT-PCR and multiple PCR is the annealing temperature. In MT-PCR this is decreased with each cycle. The first cycle (initial) annealing temperature is set 3~5°C higher than the expected temperature. Generally the higher the annealing temperature, the better the matching of the primers and templates. A slow decrease in annealing temperature makes PCR more efficient.
The precise and steady control of temperature is vital
The researchers of this MT-PCR study successfully identified 5 target DNAs, mecA, Blashv, Blactx-M, BlaTem, and Blaoxa in blood culture bottles and spike blood bottles, see Figure 1. At the same time, there was no cross-amplification and no false positive results. (Figure 2) In the experiment, the initial annealing temperature set on the thermal cycler was 66°C for 30 seconds. Researchers continued running the reaction for 20 cycles, but for each cycle the annealing temperature was lowered by 0.5°C. The precision and stability of temperature control are essential. TurboCycler thermal cycler from BlueRay Biotech is very stable and can control the temperature precisely and steadily. It has a high ramp rate which allows it to be set quickly to the specific temperature needed. It also has a sample mode which keeps the temperature difference between the sample and test block to an absolute minimum. TurboCycler is researchers’ ideal choice for MT-PCR.
Fig. 1 The MT-PCR result of samples from 12 spiked blood culture bottles. No 9~No. 13 are the control groups for the target genes. No.1~No.8 showed clear expression for the target genes in MT-PCR array without other cross-amplification.
Fig 2. The result of samples from 33 positive blood culture bottles in 4h MT-PCR array. The target genes are expressed clearly without non-target gene expression.
In the study, the target bacterial DNAs held genes for multiple antibiotic resistance. Patients infected by those bacteria need to be given the correct antibiotic treatment without delay or sepsis can result in serious consequences. This work proved that MT-PCR could quickly identify the bacteria involved.
MT-PCR can not only quickly identify the genes of several target bacteria in the samples, but also cross-amplification for non-target DNA can also be avoided. False positive results are remarkably fewer than before and the process is of great value for both clinical research and diagnostics. We expect there to be many more MT-PCR applications in the near future. It is also very important to choose a good thermal cycler for the best results from PCR.
1.Ming-Yi W, Jian-Li G, Ying-Jian C, Yu S, Mei S, Hai-Zhu L, and Cheng-Kin H. Direct Detection of mecA, blaSHV, blaCTX-M, blaTEM, and blaOXA Genes from Positive Blood Culture Bottles by Multiple-touchdown PCR Assay.
2.Henegariu O, Heerema N A, Dlouhy, S R, Vance, G H and Vogt, P H (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504-511.
POLYGEN præsenterer en række nyudviklede HPLC kolonner fra Dr. Maisch, Tyskland:
Katalog over disse Repromer kolonner med beskrivelser, applikationer, liste over retentionstid samt en krydsliste over konkurrent kolonner og tilsvarende Repromer kolonner kan hentes her i PDF format.
Tilbud, yderligere oplysninger samt priser kan indhentes her firstname.lastname@example.org
Vi tilbyder disse kolonner til meget konkurrencedygtige priser samt tilbyder afprøvning i en periode af 14 dage – uforpligtende – og kan sendes retur, såfremt kolonne ikke giver tilfredsstillende resultater.
What are preparative, semipreparative and analytical HPLC?
Please help me in understanding the exact differences between analytical HPLC, semipreparative HPLC and preparative HPLC
Analytical HPLC is usually carried out under high backpressures. For example in Reverse-Phase High Performance Liquid Chromatography by using 250 cm length x 4.6 mm Inner Diameter, and 5 micras C18 columns, and flow rates around 0.5 ml/min of solvents like Acetonitrile (low viscosity) and temperatures about 20 or 30ºC, the minimum backpressure recommended is 11 bar.
Otherwise, preparative and semipreparative chromatography are carried out under low backpressures (1-5 bar) with bigger inner diameter columns and bigger particle size (10 micras in some cases) by passing mobile phases at high flow rates (4-5 ml/min). Alternatively, you can accomplished preparative and semipreparative chromatography by using a short inner diameter and small size particle (5 micras) by passing a very small flow rate (about 0.05 ml/min) mobile phases with very low viscosity (hexane for example) in the aim of keeping a very low backpressure and saving solvents.
In addition, the most important difference between preparative/semipreparative and analytical HPLC is the purpose. Whereas the first one is designed for obtaining the fractionation of groups of compounds (for example fractionation of PAHs and oxy-PAHs) usually as a function of their polarity, analytical HPLC is designed for separation of individual compounds as a function of their medium polarizabilities.
Maybe these two articles can help understanding the mechanisms of both methodologies.
Vores UK leverandør har et stort program af centrifuger, herunder indenfor hospitals brug:
Clinical (hospital) centrifuge – se brochure over hele programmet PrO-Hospital Centrifuges-brochure
Cytology – se brochure over hele programmet PrO-CytologyCell Washer (CW) – se brochure over hele programmet PrO-CW-brochureProtein Rich Plasma (PRP) – se brochure over hele programmet PrO-CW-brochure
TurboCycler 2 gradient contra SensoQuest LabCycler Gradient
Da vi er blevet distributør af TurboCycler – både af model 2 gradient og Lite gradient – har vi foretaget en sammenligning med en af de mest udbredte ThermalCycler i Danmark, nemlig LabCycler gradient fra firmaet SensoQuest imod vores TurboCycler 2 gradient.
Protein Analysis with Size Exclusion Chromatography
Gel permeation chromatography/size-exclusion chromatography (GPC/SEC) is the standard method to separate samples by molecular size. In protein analysis, size-exclusion chromatography is either applied to detect and quantify aggregation, or to measure the complete molar mass distribution. However, method development is not trivial and the choice of suitable detection options is crucial.
Tips & Tricks – 04.2017 – D. Held and T. Hofe