How can a 'single primer PCR' be possible?

Posted by Jack on December 13, 2022
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    Hi there! I'm wondering how you can do single primer PCR. Can you tell me about it?

    I want to amplify a part of my genome for sequencing.

    You are probably here because you want to amplify a part of your genome for sequencing. In this case, it is necessary to use a protocol called single primer PCR (or 'primer walking' as it is sometimes called). This method of PCR uses only one primer and therefore can only be used on small fragments of DNA.

    I have sequences of one of the fragments already (using other primers).

    If you have sequencing data of your gene, you can use this to find a suitable primer for it. For example, if I were working on the human ABO gene and wanted to study its exon structure, I would first use BLAST to search for other ABO-related sequences. Once I found a similar sequence (say FJ566180), I could look at what primers they used in their experiment and see if any of them might work as primers for my own project.

    How can I design a primer? It should be a single primer PCR.

    To design a single-primer PCR, we need to meet some criteria for the primer:

    • The length of it must be 20-25 bases. If it's too long or too short, it won't work properly.
    • It should match the sequence of interest at both ends (i.e., 5’ and 3’). If you want to amplify an intron region of DNA, you will need two primers: one that matches one side and another one that matches the other side (5’UTR). In order for your PCR reaction to work correctly, both sides have to be amplified in parallel so they can produce a full length product afterwards.
    • The GC content should be 40-60%

    Why can't I amplify a longer sequence?

    You might be wondering why you can't amplify a longer sequence. The answer is that single primer PCR is a very specific technique, and you have to follow the rules. In order to make sure that you get the right length of DNA to amplify, you need to design your primers correctly. You also need to use a special technique called thermal cycling in order to get good results from this kind of experiment.

    Single primer PCRs are possible if you do them right.

    The key to performing a single primer PCR is to use primers that are short and have high annealing temperature (the temperature at which the primer will bind to the target DNA). You also need to use a large concentration of dNTPs and MgCl2 in your PCR reaction.

    The following figure shows an example of a single primer PCR.

    The following figure shows an example of a single primer PCR.

    The above diagram illustrates how a single primer can be used in PCR to create two complementary strands of DNA. The forward and reverse primers are shown as red and blue arrows, respectively. They bind to the DNA template strand (blue), which is complementary to them at each end. When you heat up the reaction mixture for a few seconds, the primers become covalently-closed together with their complimentary sequences on opposite ends of the template strand (shown in green). This forms a looped structure called a secondary structure that is held together by hydrogen bonds between bases on each strand; this is known as an "annealing" reaction because it literally attaches different pieces together through base pairing interactions!

    Is this possible?

    Yes, it is possible. However, this is not recommended for a number of reasons. First, the results are unpredictable. The product may be shorter than expected and may be a mixture of fragments rather than a single amplified product. Second, some primers may have been contaminated with primer dimers during synthesis or in storage (these are larger molecules that will not give rise to an amplifiable product). This means that even if you use only one primer pair per reaction tube and get amplification from your gel electrophoresis results, you might still end up with many different products instead of a single band at the size predicted by your initial sequencing run.

    Usually, PCR uses two primers; one at each end of the fragment to be amplified.

    The standard PCR protocol uses two primers to amplify the DNA. One of these primers is located at one end of the fragment to be amplified, and the other is located at the other end. The polymerase uses each primer to initiate complementary DNA synthesis in an enzymatic process that can be repeated many times to produce many copies of the DNA fragment.

    These primers are usually designed based on the DNA sequence knowledge.

    In short, the primers are designed based on the sequence of the target DNA. They are designed to match a specific part of the DNA fragment, which is known as "primer binding site". Primer binding sites must be complementary to each other for every primer pair in order to form a complete double stranded DNA at their ends.

    The polymerase uses these primers to initiate complementary DNA synthesis.

    The polymerase enzyme uses these primers to initiate complementary DNA synthesis, which is the process of building new double-stranded DNA from single stranded templates. The primer is the starting point for this reaction.

    A PCR can be run with only one primer, but the results are unpredictable.

    A PCR can be run with only one primer, but the results are unpredictable. In this case, only one primer is used in the PCR. The other strand of DNA is synthesized by a complementary DNA strand that forms from a template and some primers (which have been added before the reaction). For example, let's say you want to amplify an 18 base pair region of human genomic DNA from p53 exon 1:

    • Design a primer based on your knowledge of the p53 gene sequence and its position within exon 1
    • Add that primer to your reaction mix along with template DNA and dNTPs; then run it as usual


    I hope this post has helped you to understand why single primer PCRs are possible and how they work. I wanted to write it because I have found that there are many misconceptions about the technique, especially online. If you have any questions or comments please leave them below!

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