When an architect wants to make perfect curves on a design, he uses a template. Your body uses template DNA to make a perfect copy of a protein. Scientists also use template DNA to make copies of a small sample of DNA using PCR.
What is Template DNA
DNA exists inside the nucleus as a double-stranded molecule.
Some areas of DNA contain genes while others control gene expression. In areas where there is genetic information (a gene), one strand is the coding strand and the other is the non-coding strand. The coding strand is the non-template strand, i.e.
, it is not copied. The non-coding strand is actually the template.
Template DNA and Transcription
Your DNA is comprised of genetic information. This information encodes the instructions for all cellular functions.
In order for your cells to function properly, this information has to be transferred from the nucleus (home of DNA) to the cytoplasm. Since DNA can’t leave the nucleus, you must make a copy of it. This is called gene expression.The first step of gene expression is transcription. In this process a gene (DNA) is copied into RNA by the enzyme RNA polymerase (RNAP in the diagram). The resulting RNA can be kept as is and used as an RNA gene product such as ribosomal RNA.
It can also be a template for protein production. If the RNA will be used to produce a protein it is called messenger RNA.We just discussed an overall view of transcription. Now let’s look a little deeper. When a DNA sequence is read by an RNA polymerase, a complementary and antiparallel RNA strand is produced.
What do we mean by complementary and antiparallel? We mean that the sequence of nucleotides in the template strand is the match (complement) to the template strand. However, they run in opposite directions, and are thus ‘antiparallel.’The copy of the template strand is read by ribosomes, which then produce a protein via translation. So, why do we use the non-coding strand as our template? Why do we call the coding strand the coding strand if we don’t use it to code for a gene product?We want the protein to be a complementary AND parallel copy of our gene. Thus, we copy the template DNA because it is the complementary but antiparallel version of the coding DNA (the gene itself).
This produces an mRNA that is complementary AND parallel to the gene. If we copied the gene itself, then the resulting mRNA would be complimentary BUT antiparallel to the gene.
Template DNA and PCR
PCR (polymerase chain reaction) is a technique in molecular biology. It is used to amplify sequences of DNA.
It is a powerful tool that can take a few copies of a gene and generate thousands to millions of copies. This technique was made famous during the OJ Simpson trial. It is what was used by forensic scientists to amplify the DNA in the blood samples found at the crime scene.The method relies on cycles of repeated heating and cooling of a chemical mixture known as thermocycling. The mixture is made of DNA, enzymes, primers, and nucleotides, and is a tiny volume. The starter DNA and all DNA generated during the PCR process is used as template DNA.
This image shows how small the reaction tubes are in PCR.
The reason for thermocycling is that in PCR, the different processes require different temperatures. The first step is to separate (melt) the double stranded DNA. This requires a high temperature, about 94-96 ;C.
After melting the DNA, the temperature is dropped to between 50-65 ;C, which allows primers to bind (anneal) to the DNA template. The temperature is not low enough for the DNA to re-anneal to itself. This is because primers are short DNA fragments containing sequences complementary to the target region on the template DNA.The temperature is then raised to 75-80 ;C, which allows the DNA replication machinery (DNA polymerase) to bind to the primer-template DNA and begin copying the template DNA. This is done by DNA polymerase reading the template and adding nucleotides one at a time. After a short period of time, usually about 2 minutes, the mixture is heated again to 94-96 ;C.The process is repeated several times (cycles).
The minimum number of cycles is 25; however, 35 to 40 cycles is common. After all cycles have finished, the temperature is brought to 70-74 ;C. This gives DNA polymerase a final chance to finish elongating any incomplete DNA copies. This figure summarizes PCR.
Let’s review! In areas of DNA where there is genetic information, a gene, one strand is the coding strand and the other is the non-coding strand.
The coding strand is the non-template strand, i.e., it is not copied. The non-coding strand is actually the template.
The copy of the template strand is read by ribosomes, which then produce a protein via translation. During transcription, we use the non-coding strand as our template because we want the protein to be complementary AND parallel copy of our gene.Using a technique called PCR or polymerase chain reaction, which relies on a process called thermocycling, we can amplify sequences of DNA.