Primers and Probes
A primer is a strand of DNA bases that enables DNA to be replicated, primers basically “jumpstart” the DNA
replication process. Primers are used primarily in Polymerase Chain Reaction, a process which multiplies DNA to
make millions of copies. Each primer is single stranded DNA and is designed to match a specific piece of template
DNA. The specificity arises from the fact that each DNA base can only pair with one other DNA base: that is adenine
(A) pairs only with thymine (T) in DNA and uracil (U) in RNA, and guanine (G) pairs only with cytosine (C). In order for
copies to be made, the primer must bind to the right piece of DNA and the bases must match. If the matching occurs,
then DNA polymerase (the enzyme that copies the DNA) can bind and amplify the DNA. If the primer does not match
the DNA sequence, then the DNA polymerase cannot bind and no copies will be made. To get exactly the right order
of A, G, T and C, we can order primers from companies that can string together the sequence we want.
Source: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A772
In the figure above, a strand of template DNA to be copied has a primer (blue) sit down on it. The primer can
sit down because the GAGA matches the CUCU. This lets DNA polymerase assemble free nucleotides (A,C,T,G) in an
order that is the complement to the starting strand, making new DNA (tan). Primers are specific but sometimes a
repeating sequence will appear in the template DNA more than once, such as a GAGA sequence. If this happens,
then it is called a degenerate primer and you may need to increase the primer length to decrease the chances of
having the same sequence elsewhere in the DNA. For example, you could take the above primer, CUCU and extend it
to make the primer CUCUAAC.
Probes - Probes are primers with an addition on one group that allows us to see the primer. This change can be a
molecule that fluoresces and shines when hit with light, or it can be an attachment to a colored bead, for example.
This then allows us to visualize when a probe (primer with a colored bead attached to it) attaches to DNA. Since we
designed the probe, we can tell what the DNA sequence is at that particular point in the DNA.
Questions:
1.
2.
3.
4.
5.
What is the main function of primers?
What is a degenerate primer?
Where do we get primers?
What is the purpose of a probe?
What do you need to keep in mind when designing a primer?
References
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A772
Probes and Primers in the GM Foods experiment - In this experiment, soy has DNA sequences which code for
different genes. One gene, lectin, is in all soy products, and another gene, the "Roundup Ready CP4-EPSPS precursor
gene” is a synthetic insert that is only found in transgenic or modified soy. Since the gene sequences are known, we
can purchase primers which will match the gene and allow us to copy it with polymerase chain reaction (PCR). Once
we have many copies, we can create probes to see if either the “endogenous” lectin gene is present only or if bot the
endogenous lectin gene and the transgenic Roundup Ready gene is also present. We create a primer based on the
DNA sequence and attach it to a red bead which will show up on the biosensor – thus making it a probe. When we
add our probe to our copies of lectin gene, the probe should stick (show red bead) to it because that gene is present
in all soy. When we add the roundup probe to our copies of the roundup ready gene, the roundup probes will only
stick (show red bead) to the soy which has this modification. This allows us to see which soy products have
modifications by comparing which samples have red beads stick and which do not.
Probes and primers in the maternal ancestry experiment – In the maternal ancestry experiment, we have designed
primers that match the mitochondrial DNA sequence on both sides of each of the 6 SNPs, so we can amplify those 6
sections of DNA. We use a combination of a common probe and a probe specific to each possible SNP to test which
SNP we have.
Questions:
1.
2.
3.
4.
5.
What is the main function of primers?
What is a degenerate primer?
Where do we get primers?
What is the purpose of a probe?
What do you need to keep in mind when designing a primer?
References
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A772