I am designing a Taqman PCR. However, I only can find a small continue specific fragment (9bp) in the target region. How should I design primers and probe?
1. place the 9 bp in either forward primer or reverse primer?
2. place the 9 bp in the probe?
3. place half (~3 bp) in either forward or reverse primer and place the other half (~3 bp) in the probe?
Dear Zhenyu Shen
Primer designing becomes very easy with a clear understanding of basics of DNA replication (After all we are designing primers to replicate the DNA in a PCR). Key things to remember from molecular biology 101 are:
1. New strand synthesis always occur in 5' to 3' direction.
2. The two DNA strands are always complementary to each other.
3. The terminologies like sense strand/coding strand/non-template strand or antisense strand/non-coding strand/template strand comes handy too.
(If the given DNA molecule is double stranded, the strand that runs in 5' to 3' direction is referred to as sense strand/coding strand/non-template strand. This is strand with the sequence same as the mRNA and hence the nomenclature. Similarly, the strand that runs in 3' to 5' direction is referred to as an antisense strand/non-coding strand/template strand. This is the strand that is usually transcribed into the mRNA)
4. Finally, the writing convention, if the directionality of a DNA molecule is not specified, as a convention, in a double stranded DNA the top strand runs in 5' to 3' direction whereas the bottom strand runs in 3' to 5' direction. If only a single strand of DNA is given, it is safe to assume that it runs in 5' to 3' direction.
Having straightened out all this, primer design is a piece of cake. To design the Forward Primer, use the first 15-20 bases of the template strand and to design the Reverse Primer, use the last 15-20 bases of the template and make a reverse compliment (that is take the last 15-20 bases, write its complimentary sequence and then read it in the reverse direction).
By doing this you will have a set of your forward and reverse primers that could be used to amplify the DNA in a PCR. Both these primers will have a 5' to 3' directionality. It is critical to check the parameters like length, melting temperature and GC content of this primer pair for a successful PCR reaction. The primer pair should not be very different in their melting temperatures (usually within 5 degrees of each other). The primers should not be too high or low in their GC content and its always a good idea to have a G or C at the 3' end of the primer, to ensure tight binding. Finally restriction sites or homologous sequences for cloning, could be added at the 5' end. Having a couple of extra bases along with the restriction site helps to ensure proper digestion..
Regards
Dear Zhenyu Shen
Primer designing becomes very easy with a clear understanding of basics of DNA replication (After all we are designing primers to replicate the DNA in a PCR). Key things to remember from molecular biology 101 are:
1. New strand synthesis always occur in 5' to 3' direction.
2. The two DNA strands are always complementary to each other.
3. The terminologies like sense strand/coding strand/non-template strand or antisense strand/non-coding strand/template strand comes handy too.
(If the given DNA molecule is double stranded, the strand that runs in 5' to 3' direction is referred to as sense strand/coding strand/non-template strand. This is strand with the sequence same as the mRNA and hence the nomenclature. Similarly, the strand that runs in 3' to 5' direction is referred to as an antisense strand/non-coding strand/template strand. This is the strand that is usually transcribed into the mRNA)
4. Finally, the writing convention, if the directionality of a DNA molecule is not specified, as a convention, in a double stranded DNA the top strand runs in 5' to 3' direction whereas the bottom strand runs in 3' to 5' direction. If only a single strand of DNA is given, it is safe to assume that it runs in 5' to 3' direction.
Having straightened out all this, primer design is a piece of cake. To design the Forward Primer, use the first 15-20 bases of the template strand and to design the Reverse Primer, use the last 15-20 bases of the template and make a reverse compliment (that is take the last 15-20 bases, write its complimentary sequence and then read it in the reverse direction).
By doing this you will have a set of your forward and reverse primers that could be used to amplify the DNA in a PCR. Both these primers will have a 5' to 3' directionality. It is critical to check the parameters like length, melting temperature and GC content of this primer pair for a successful PCR reaction. The primer pair should not be very different in their melting temperatures (usually within 5 degrees of each other). The primers should not be too high or low in their GC content and its always a good idea to have a G or C at the 3' end of the primer, to ensure tight binding. Finally restriction sites or homologous sequences for cloning, could be added at the 5' end. Having a couple of extra bases along with the restriction site helps to ensure proper digestion..
Regards
I would like to add a few more things.Depends on what the primers are for. If they’re for amplifying a chunk of DNA, you want one primer to start the sense strand and another to start the anti-sense strand. They should have a pretty similar melting temperature (Tm), so they’ll work together well, and they probably shouldn’t be too far apart, so the piece of DNA they make can be made fairly quickly. We normally look for an amplified piece of something like 300–700 base pairs for regular PCR. Different purposes will allow (or require) different sizes. As a rough guide, you probably want primers of this kind to be about 20 base pairs or so, although that can vary depending on the G-C content and how high (or low) you want your Tm to be. If you want to use the primers to introduce a mutation in the piece you’re amplifying, you’ll want to read the protocol to ensure you get something that will work the way you want it to.
If your primer is for sequencing, that’s a different kettle of wax all together, and you’d want to look into the various methods of sequencing to see how to go about designing a primer for one of those purposes.
Hi,
I am sorry. The question is not clear.
The purpose of the PCR is differentiate one genotype from the others. The target only has a small specific continue fragment (9bp). The other areas in the target have identical sequences with other genotypes, My question is where should I place the specific fragment (in one primer, in the probe, or in one primer and the probe ?) if I have to use PCR methods..
Muhammed H Musleh and Mushtak T. S. Al-Oukaili---- nicely explained, benefited from these answers. Thanks and regards
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