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. What is the main function of primers?

 2. What is a degenerate primer?
3. Where do we get primers?
4. What is the purpose of a probe?
5. 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. What is the main function of primers?

2. What is a degenerate primer?
3. Where do we get primers?
4. What is the purpose of a probe?
5. 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