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How do they sequence a gene Part 1: PCR

In response to my discussion about genetic testing, I’ve been asked, “How do they do that?” They do it by sequencing the DNA. In my multi-part answer to this question, I’ll begin with the first step of that process: PCR.
PCR stands for “Polymerase Chain Reaction”. That might be a bit of an exaggeration, as it sounds like molecular biology gone wild. It is actually a very controlled and fascinating technique — quite possibly the most significant innovation in molecular biology in the past twenty-five years.
But first, lets cover some of the principle features of DNA: it is double stranded, composed of a repeating molecular letters that are complimentary and the two strands run antiparallel to each other.
What am I talking about????
Well, as I’m sure you’ve heard, DNA is a double-helix. The double-helix made up of a repeating sequence of a class of molecule known as a nucleic acid. There are four kinds nucleic acids that make up the helix. They are abbreviated as A, T, G and C. Furthermore, the nucleic acids of one strand are complimentary to the molecules in the other strand: An A in one strand is paired with a T in the other strand, and a C in one is paired with a G in the other. The cells of your body read the sequence of nucleic acids much like you read a reference book: you go to the chapter and begin reading the letters from left to right. DNA has a “left to right” direction too (but they call it “five prime to three prime”). DNA is antiparallel because when one strand is going left to right (five prime to three prime), its complimentary strand is going right to left (three prime to five prime). I’ve illustrated this in the diagram: The bottom strand’s letters are upside down.
To begin a PCR reaction, you need two important ingredients: A small amount of template DNA and primers. Template DNA is extracted from the blood of the patient. Each blood cell has the patient’s complete genomic DNA in its nucleus. The primers are short, single-stranded DNA molecules that are manufactured using modern chemistry techniques. Primers are designed to be complimentary to the template DNA. Furthermore, there are two primers that mark where to begin and where to end the manufacturing of DNA through PCR.
Okay, now on to the reaction itself. Here is a diagram illustrating the steps of PCR:


PCR (right click/control click and choose “Zoom In” for more detail)

PCR has three steps: denaturing, annealing and extending.
In the first step, heat is applied to the template DNA to pull the two strands apart to create single stranded template DNA. This process is known as denaturing. Next, the sample is allowed to cool with an excess of primer DNA in the test tube. The primer DNA is perfectly complimentary to the ends of the template DNA, thus it forms short regions of double-stranded DNA with the template. This step is called annealing. Finally some DNA Polymerase is added along with all the necessary nucleic acids, and the remaining single-stranded section of DNA is converted to double stranded as the primer is extended with complimentary bases. From one molecule of DNA, two have been made.
The “chain reaction” occurs when this process is repeated over and over, resulting in first 2, then 4, 8, 16… 2n copies! Most people stop around 15-20 cycles, but some people who are very daring somtimes go up to 30 or even 40 cycles.
Thus from the small amount of DNA that was extracted from the patients blood, a large quantity has been “amplified” using PCR. The amplified DNA will be used in the sequencing reaction… stay tuned!

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