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Polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR)

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Polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR)

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High Yield Notes
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Polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR)

14 flashcards
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USMLE® Step 1 style questions USMLE

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A 6 month-old male is thought to have sickle cell anemia, and as his pediatrician you want to sequence the gene that may be mutated, causing this disease. You decide to sequence the gene for the beta subunit of hemoglobin. In order to do this you must perform polymerase chain reaction (PCR). You need to know which of the following in order to amplify (PCR) the area of interest of his DNA?

Transcript

Our DNA contains loads of information, neatly stacked to insanely small sizes, able to fit within a cell nucleus.

A single DNA molecule has two strands, which wrap one around one another to form a double helix.

Each single strand of DNA is composed of a sequence of four types of nucleotides - which are the individual letters or building blocks of DNA.

Nucleotides of DNA are made up of a sugar - deoxyribose, a phosphate, and one of the four nucleobases - adenine, cytosine, guanine, and thymine - or A, C, G, T for short.

The nucleotides on one strand form hydrogen bonds to complementary nucleotides on the other strand; specifically, A bonds with T via two hydrogen bonds, and C bonds with G, via three hydrogen bonds.

Additionally, the two DNA strands also have a “direction” - meaning, one of them runs from the 3’ end to the 5’ end, while the other one runs from the 5’ end to the 3’ end.

Kinda like two snakes coiled up together, but facing in different directions. Every single protein of our body is encoded through combinations of just four nucleotides.

When there are errors in our genetic information, diseases occur. And let’s be honest, we were always interested in knowing what was written in our DNA.

Polymerase chain reaction, or PCR for short, is a technique used in molecular biology to amplify a segment of DNA. Let’s take a step back. A single copy of DNA is not very much DNA.

So to work with DNA, we basically make lots and lots of lots of copies of it, so that it’s easier to analyze. For example, if we want to visualize it, we can use a technique called gel electrophoresis.

PCR is based on DNA replication, a process that our cells normally use to duplicate their genetic material before dividing in two identical daughter cells.

So first of all, we’re going to need a machine called a thermal cycler - that’s where the PCR magic happens.

You can think of it like a cauldron filled with a solution, where genetic wizards add the ingredients.

The ingredients are the DNA that we wish to multiply, an enzyme called Taq polymerase, specific primers, that bind to the DNA, and a mixture of free nucleotides - A, T, C and G. Throw everything in the thermal cycler, wave your magic wand, and the process begins.

So, let’s say that we have a long, double stranded DNA molecule, and we’re interested in the highlighted part. These two strands would be the template strands.

5’ - T T C A G G T C A C A G T C C T G T A T G C C T A T G T C C- 3’
3’ - A A G T C C A G T G T C A G G A C A T A C G G A T A C A G G- 5’

The first step of PCR is denaturation - meaning that we heat up our ingredients to exactly 96 degrees Celsius - almost as hot as boiling water.

This breaks open all bonds between the two strands of DNA, so that they can separate from one another.

The second step is called annealing. Here’s where we need primers, and for our highlighted sequence, the primers would look like this:

<----3’- C G G A T A C - 5’ 5’ - A G G T C A C - 3’ --->

During the annealing step, we cool everything down to around 55 degrees Celsius, and this allows the primers to bind to their complementary sequences on the single stranded DNA.