Single-molecule PCR

Summary

Single-molecule PCR, also known as homo-primer PCR, is a PCR technique in which a single DNA molecule is used as a template and the number of cycles is unlimited.The development of PCR technology has become quite mature nowadays, and has given rise to many new techniques, such as RT-PCR, RAPD, AFLP, reverse PCR, Nested PCR, hot-start PCR, colony PCR and real-time PCR, etc. Generally speaking, in order to amplify the target DNA efficiently, a larger amount of template is added in the reaction system, and a smaller number of cycles is used in the reaction procedure. Generally speaking, in order to amplify the target DNA efficiently, more template is added to the reaction system and fewer cycles are used in the reaction program. If the amount of template is too small, the amplification efficiency will be low and it will be difficult to amplify the target DNA efficiently, and if the number of cycles is too large, the proportion of non-specific amplification will increase. Therefore, the amount of template is usually limited to the pg or ng level, and the number of cycles is usually less than 40. However, theoretically, the amount of template can be as low as one DNA molecule, and the number of cycles can be unlimited. From this theory, a new PCR technique, single-molecule PCR, has recently been developed. Currently, there is one main method used for single-molecule PCR: single-molecule PCR.

Principle

The basic principle of single-molecule PCR is that single-molecule PCR is derived from conventional PCR, and its principle is no different from that of conventional PCR, the only difference is that the amount of template is as low as a few or even a single molecule, so the number of cycles needs to be higher than that of the conventional PCR. Therefore, in order to ensure the success of the test, the quality of the template, primer design, selection of DNA polymerase, reaction conditions and specific operations are quite different from those of conventional PCR.

Appliance

The common application fields of single-molecule PCR are as follows:

1. Constructing protein libraries

Under the action of high-fidelity DNA polymerase, the SIMPLEX system, which is formed by combining SM-PCR and in vitro cell-free expression system, has the advantages of high expression and rapid construction of protein libraries, which can make up for a series of shortcomings of the conventional protein display technology and extend the application scope of the protein display technology, especially for the expression of toxic proteins, which can obtain protein libraries of different sizes by changing the number of DNA templates. The SIMPLEX system has the advantages of high expression and rapid construction of protein libraries. The Laboratory of Molecular Biology, Nagoya University has successfully constructed protein libraries of anti-human serum protein single-chain antibody, lipase and other proteins by using SIMPLEX system.

2.In vitro directed evolution

In vitro directed evolution is a technique to irrationally modify proteins or DNA molecules. At present, this technology is just emerging, but it is developing rapidly. SM-PCR, under the joint action of low-fidelity TaqDNA polymerase and other mutation-causing factors, is very easy to cause DNA mutation, and thus constructs DNA libraries containing a large number of random mutations. When using Taq polymerase with a standard mismatch rate of 0.8X 10-5 bases per cycle, SM-PCR with deviation from the reaction conditions can get 2.5X10-5 bases per cycle. obtain a mismatch frequency of 2.5X10-5 bases when using a standard Taq polymerase of 0.8X10-5 bases per cycle. Therefore, it has a broad application prospect in the field of in vitro directed evolution.

3. Genome Sequencing 

Because SM-PCR can produce mutation-free DNA under the action of high-fidelity DNA polymerase (e.g., Pfu DNA polymerase), it can be widely used for sequencing various genes. Sequencing results of a 410bp readable sequence after SM-PCR show that the accuracy of PCR can reach 99.3%. This is similar to the results of PCR with a DNA template of 100. At present, although the Human Genome Project has been completed, its sketch covers only 85%, and there are still many animal, plant and microbial genome projects in progress, which have greatly contributed to the rapid development of SM-PCR technology.

4. Forensic Identification

Currently, SM-PCR is being used more and more widely in the field of forensic science, such as gender identification, individual identification, kinship identification, and species identification, etc. With the proportion of intelligent crimes increasing year by year, SM-PCR can be used to identify the sexes of individuals and their families, as well as the number of individuals and their families in a particular area. As the proportion of intelligent crimes increases year by year, and the suspects' awareness of anti-investigation increases, the biological evidence left at the crime scene will be scarce, and forensic scientists have to obtain DNA polymorphic information from trace or ultra-trace biological samples (e.g., eyeglasses, earphones, toothbrushes, chopsticks, sweat, skin fragments extracted from fingerprints, oral epithelium, etc.) as evidence, and the conventional PCR methods are not always suitable for this kind of identification, and SM-PCR is not suitable for this kind of identification due to its template requirement. However, SM-PCR can fully meet the needs of forensic evidence identification due to its extremely low template requirement. In addition, in the field of forensic science, there is still no practical and effective method to solve the problems of separating and identifying the sperms left by different male individuals and separating and identifying the mixed blood cells of different individuals in gang-rape cases, and we can only compare the methods, which reduces the accuracy of individual identification. The combination of single-cell laser capture technology and SM-PCR technology is undoubtedly an ideal means to solve these problems, and SM-PCR technology has great potential for research and application in the field of forensic science.

Operation method

Single-molecule PCR

Principle

The basic principle of single-molecule PCR is that single-molecule PCR is derived from conventional PCR, and its principle is no different from that of conventional PCR, the only difference is that the amount of template is as low as a few or even a single molecule, so the number of cycles needs to be higher than that of the conventional PCR. Therefore, in order to ensure the success of the test, the quality of the template, primer design, selection of DNA polymerase, reaction conditions and specific operations are quite different from those of conventional PCR.

Materials and Instruments

Equipment: PCR instrument.
Reagents: 10 X PCR buffer (0.1 mol/L Tris-Cl pH 8.8, 15 mmol/L MgCl
2
, 0.5 mol/L KC1 and 1 % TritonX100), DNA template to be tested, 2.5 mmol/L dNTP, l0pmol upstream primer LDS, 10pmol downstream primer LDK, TaqDNA polymerase.

Move

The basic process of single molecule PCR can be divided into the following steps:

1. Reaction system

First, the purified DNA is diluted to a final concentration of 1 (or 5 or 10) molecule/μl with TE solution containing lg/L blue dextran 2000 or lng/L tRNA. Next, each component is added separately according to the reaction system described below. 2.

2. Reaction Procedure

Caveat

SM-PCR is developed based on the principle of conventional PCR technology, which includes 3 basic steps of denaturation, denaturation and extension, but it is quite different from conventional PCR in terms of the number of templates, primer design, selection of DNA polymerase and reaction conditions.1. Preparation of templateSM-PCR is an amplification reaction in which the DNA template is diluted to extreme conditions, so the quality of the template directly determines the success of the experiment and the amplification efficiency. Purified DNA is diluted to a final concentration of 1 (or 5 or 10) molecule/μl with TE solution containing lg/L Blue Dextran 2000 or lng/L tRNA. The purpose of blue dextran 2000 or tRNA is to prevent DNA adsorption on the wall of the tube to ensure proper denaturation of the template, annealing of the primers, and extension of the strand.

2. Primer designThe amount of template in the SM-PCR reaction mixture is very low, and if there are a few paired sequences between the primers, it is very easy to form a dimer under multiple cycles of amplification. Therefore, when designing primers, the (G+C) content and Tm value should be strictly controlled, and the presence of paired sequences between primers should be avoided as much as possible. In 2000, Nakano et al. modified the template to obtain a template with the same end, and used a single-primer for SM-PCR (also known as homo-primer PCR). Since this single primer was artificially introduced, it can be fully optimized, thus effectively avoiding the generation of primer dimers and improving the success rate of the experiment.

3. Selection of DNA polymeraseDNA polymerase is the most important part of the PCR reaction, and its fidelity and thermal stability will determine whether it can amplify the ideal product. In SM-PCR, the number of templates is small, the number of cycles is large, and the loss of dNTP is large. Therefore, under the action of DNA polymerase with low fidelity and poor thermal stability, mutation is easily generated. Therefore, the choice of DNA polymerase depends entirely on the need to generate mutations. For genome sequencing and forensic identification, a high-fidelity PufDNA polymerase with an error incorporation rate of 7X10-7 polymorphs/bp is often used to produce large quantities of highly uniform PCR products that are free of mutations. A 500-bp fragment can be amplified one million times at the same experimental efficiency with an error rate of only 0.7%.

4. Reaction conditionsThe main differences between SM-PCR and conventional PCR are as follows: ① The denaturation temperature of SM-PCR is higher than that of conventional PCR (94°C), and its denaturation time is shorter than that of conventional PCR (40-60 s) (5-15 s), and its annealing time and extension time are also shortened accordingly; ② The number of cycles of conventional PCR is 35-40 times, but the number of cycles of SM-PCR is 60-80 times. (ii) The number of conventional PCR cycles is 35~40 times, but the number of SM-PCR cycles reaches 60~80 times, and the total reaction time is as much as 3~5 h. Therefore, it is necessary to choose a DNA polymerase with good thermal stability.

5. Prevention of contaminationContamination has a greater impact on SM-PCR than on other PCR derivatization techniques. Since the number of templates in SM-PCR is very small, a small amount of contamination can cause errors in the experiment. A small amount of contamination can lead to errors or even failure of the entire experiment. During sample handling, micropipettes, air, etc. can easily cause cross-contamination between samples. In addition, the preparation of reagents is also very easy to be contaminated and lead to the contamination of SM-PCR ° Therefore, we should pay attention to set up a negative control, and at the same time, pay attention to the use of effective precautions, such as strict aseptic operation, reagents through high temperature and high pressure sterilization and dispensing, and so on.

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