In this figure, the primers are depicted as arrows. This is because, as explained below, DNA molecules are asymmetrical, a property which is important in the processes of DNA replication and transcription.
To put it simply, the process of DNA synthesis can occur in only one direction (shown by the arrows). Thus, two primers must anneal to the template DNA on opposite strands, and in opposite orientations such that they face each other, in order to achieve synthesis of both strands of the DNA segment which lies between the two primers.
In the figure above, primers 2 and 5 anneal to opposite strands and face each other, leading to the synthesis of product A.
Likewise, primers 3 and 6 anneal to opposite strands and face each other, leading to the synthesis of product B.
There will be no amplification of the DNA which lies between primers 3 and 5.
For more details, read on...
If you look closely at the chemical structure of a DNA molecule, you will see that the 5' end (at the top of the figure) of a DNA molecule is characterized by a free phosphate (P) chemical group.
If you look at the 3' end (at the bottom of the figure), you will see that it is characterized by a free hydroxyl (OH) chemical group.
By convention, when you see a DNA sequence written from left to right, we say that it is written from the 5' to 3' end (this is the "forward direction"):
A double-stranded DNA molecule consists of two asymmetrical single-stranded DNA molecules.
And since we know that A always base-pairs with T and that C always base-pairs with G, then we know that:
It is important to realize that when two single-stranded molecules anneal to form one double-stranded DNA molecule, the two single-stranded molecules are pointed in opposite directions:
The DNA template in a PCR reaction is usually a double-stranded DNA molecule, which consists of a top strand, in which the 5' --> 3' orientation goes from left to right; and the bottom strand, in which the 5' --> 3' orientation goes from right to left:
The first step in DNA replication or synthesis is melting (or pulling apart) the 2 strands of a double-stranded DNA template molecule.
Next, the primers anneal to a complementary sequence on the DNA template molecules which are now single-stranded .
The PCR primer will anneal to the DNA only if its orientation is opposite to the orientation of the DNA strand to which its sequence is complementary.
Once a primer has annealed to the DNA template, an enzyme (DNA polymerase) can add the appropriate nucleotide subunits to synthesize the new DNA strand.
To see an illustration of DNA synthesis, click
The direction of the DNA synthesis is determined by the sequence of the primer. DNA synthesis will begin only from one end (the 3' end) of the primer.
The synthesis of a new DNA strand during replication (or PCR) can only occur in one direction:
5' --> 3'.
Since the two strands of DNA in a double-stranded DNA molecule point in opposite directions, replication of the two strands occurs in opposite directions.
DNA synthesis occurs only in a 5' to 3' (5'-->3') direction.
5' --> 3' synthesis means that DNA chain is lengthened as the phosphate group at the 5' carbon of the sugar of one nucleotide subunit is linked to the hydroxyl group of the 3' carbon of the sugar of the next nucleotide as shown here:
Therefore, DNA synthesis occurs only through addition of nucleotide subunits to the 3' end of a DNA molecule.
Any questions or comments? Send them to harlocker@mbcl.rutgers.edu (Sue Harlocker)