Question

1. What are some drawbacks to PCR?

2. Why is it necessary to clean up PCR prodcuts prior to sequencing?

Answer 1

1) However, there are also disadvantages to the polymerase chain reaction. Although it has high specificity, this only works under certain conditions. The length of the target DNA sequence is a factor in the level of specificity. If the target sequence is too long then it is difficult to ensure that the target sequence will be copied in its entirety and accurately. Also if a primer sequence appears more than once in a DNA molecule then the primer may attach to more than one area, causing the experiment to copy more than one sequence or the wrong sequence completely. This can occur when certain subtypes of HIV-1 are present in the patient, causing false negative results. This is apparent in a documented case of an English tourist who contracted HIV while on holiday in Thailand. The patient displayed the symptoms associated with HIV and so was tested. HIV-1 proviral DNA PCR test was taken approximately 20 days and 26 days after infection and repeatedly gave negative results. Only 33 days after infection did the HIV-1 proviral DNA PCR test give a positive result. Due to this discovery, there has been cautious about using PCR for detection of HIV and it is recommended that any PCR results should be followed up by a conventional method of diagnosis.

First, a PCR can’t be any better than the primers. If your primers don’t pick up the target due to a mutation, that sequence will drop out. In the primer region, you can’t trust the sequence at all.

Second, PCR can mangle your sequence, particularly around repeats. It is possible to PCR both simple (such as VNTRs) and large repeats, but without care, one can get truncations or expansions which are due to PCR. Chimeric molecules can accumulate, particularly if amplifying a population of very similar molecules such as 16S RNA using consensus primers. The chimeras form from incomplete PCR products serving as primers.

Third, you are limited to the limits of PCR. PCR amplicons get increasingly tricky over a few kilobases in size. So a large target must be broken into many amplicons. There is also the workload involved in setting up an running many amplicons. Multiplexing your PCRs is possible but potentially tricky as primers may interact with each other or with other amplicons to give spurious products or reduce PCR efficiency. Overlapping amplicons are the most tricky, though a technique was recently published (and patented) for this called SLIMamp.

Fourth, PCR erases any methylation or other DNA modifications. So if you want to study those, you are out-of-luck. On the other hand, on the Oxford Nanopore platform, your base-calling accuracy will go up, as methylated nucleotides give a different signal than unmethylated ones and this interferes with base-calling.

2) You need to purify PCR products to get rid of unused primers, nucleotides, and enzymes in order to optimize the success of ligation, which will maintain and sequence the product of PCR. To purify the PCR products, size exclusion chromatography is used. The sequencing workflow usually starts with PCR also known as Polymerase Chain Reaction, where the target fragment is replicated into thousands of copies under a controlled condition using primers, DNA polymerases, and deoxynucleotides. Before the PCR product is ready for Sanger sequencing, you must do one more thing, a PCR clean up step. Because Sanger sequencing is a highly accurate technique for you to read DNA sequence base by base, it is very important to clean up your reaction mixtures so that those unincorporated primers and dNTPs won’t interfere with your results. Ethanol precipitation is the most cost effective but is also the most labor intensive. Bead or column based purification can be an effective method for many applications. However, this route tends to be a bit pricier compared to other options. This method of purification also requires transferring samples in and out of the column which can often result in the loss of some of your precious samples so this may not be the best method for those with limited starting materials. An enzymatic cleanup approach has a really simple and straightforward workflow. In fact, it’s only one single pipetting step! It is also relatively affordable compared to the bead or column based purification methods. Basically, you add an enzyme mix to your finished PCR reaction and let it sit at a certain temperature.