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Biology library

Course: Biology library > Unit 20

Lesson 3: DNA analysis methods
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Biology library
Biotechnology
DNA analysis methods
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Polymerase chain reaction (PCR)

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A technique used to amplify, or make many copies of, a specific target region of DNA.

Key points:

  • Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism).
  • PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.
  • In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.
  • PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.

What is PCR?

Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions!) of a particular region of DNA. This DNA region can be anything the experimenter is interested in. For example, it might be a gene whose function a researcher wants to understand, or a genetic marker used by forensic scientists to match crime scene DNA with suspects.
Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.
PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.

Taq polymerase

Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus).
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active around 70, °, start text, C, end text (a temperature at which a human or E. coli DNA polymerase would be nonfunctional). This heat-stability makes Taq polymerase ideal for PCR. As we'll see, high temperature is used repeatedly in PCR to denature the template DNA, or separate its strands.

PCR primers

Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis. In a PCR reaction, the experimenter determines the region of DNA that will be copied, or amplified, by the primers she or he chooses.
PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied. The primers bind to the template by complementary base pairing.
Template DNA:
5' TATCAGATCCATGGAGT...GAGTACTAGTCCTATGAGT 3' 3' ATAGTCTAGGTACCTCA...CTCATGATCAGGATACTCA 5'
Primer 1: 5' CAGATCCATGG 3' Primer 2:
When the primers are bound to the template, they can be extended by the polymerase, and the region that lies between them will get copied.
Three steps of the polymerase chain reaction are shown in the diagram. The first step has 2 parallel blue lines and each line has small, evenly spaced vertical lines extending the length of each line, the vertical lines face each other to create a structure that looks like a ladder. The image is labeled Template DNA. An arrow points to the second image with the label primers bind to template. The 2 parallel blue lines are separated; on the top blue line on the left there is a red horizontal line with an arrow pointing to the right that has 10 small vertical lines that are aligned with 10 vertical lines from the top template strand. On the bottom blue line on the right is a red horizontal line with an arrow pointing to the left. The red line has 10 small vertical lines that are aligned with 10 vertical lines from the bottom template strand. An arrow points from the second image to the third image in the diagram that is labeled Taq polymerase extends primers. On the left side of the top template strand there is a red horizontal line with 10 vertical lines joined with a gray horizontal line with 15 vertical lines and an arrow pointing to the right. The vertical lines on the red and gray line are aligned with the vertical lines on the top template strand. On the right side of the bottom template strand there is a red horizontal line with 10 vertical lines joined with a gray horizontal line with 15 vertical lines and an arrow pointing to the left. The vertical lines on the red and gray line are aligned with the vertical lines on the bottom template strand.

The steps of PCR

The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.
The basic steps are:
  1. Denaturation (96, °, start text, C, end text): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
  2. Annealing (55 - 65°, start text, C, end text): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
  3. Extension (72, °, start text, C, end text): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
A diagram of the five steps of the polymerase chain reaction are shown. In the first step, two blue parallel lines connected by small vertical lines extending from each horizontal line are shown at the top of the diagram. In the second step, an arrow labeled denaturation, 96 degrees Celsius points to the 2 parallel blue lines separated from each other. In the third step, an arrow labeled primer annealing, 55 degrees Celsius points to the 2 parallel blue lines with a small red line joined to each line. The top blue line has a red line attached on the left side of the line, and the bottom blue line has a red line attached on the right side of the line. In the fourth step, an arrow labeled primer extension, 72 degrees Celsius points to the 2 parallel blue lines, each with a small red line joined to each line and a longer gray line extending from the red line. The top blue line has a red and gray line with an arrow extending from the gray line pointing to the right, and the bottom blue line has a red and gray line with an arrow extending from the gray line to the left. In the fifth step, an arrow points from this image to a box that has 2 ladder shaped structures. In the top structure the blue line is on the top and it is connected to a small red line and gray line, and the bottom structure has a gray line and small red line on the top connected to a blue line on the bottom. The box the structures are in is labeled result after 1 cycle, number of DNA molecules doubled. From the box there is an arrow pointing upward and back to the initial blue ladder structure in the first step, with the caption on the arrow repeat 25 to 35 times.
This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours, depending on the length of the DNA region being copied. If the reaction is efficient (works well), the target region can go from just one or a few copies to billions.
That’s because it’s not just the original DNA that’s used as a template each time. Instead, the new DNA that’s made in one round can serve as a template in the next round of DNA synthesis. There are many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can roughly double in each round of cycling. This pattern of exponential growth is shown in the image below.
The diagram shows the process of DNA amplification using polymerase chain reaction through 3 cycles. An image of a ladder has 2 arrows pointing to 2 new structures and is labeled cycle 1. Both the ladders in cycle 1 have 2 arrows pointing to 2 new ladder images for a total of 4 ladder images labeled in cycle 2. Each ladder from cycle 2 has 2 arrows pointing to 2 new ladder images, all with the label cycle 3. There is a total of 8 ladders after cycle 3.

Using gel electrophoresis to visualize the results of PCR

The results of a PCR reaction are usually visualized (made visible) using gel electrophoresis. Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it separates DNA fragments according to size. A standard, or DNA ladder, is typically included so that the size of the fragments in the PCR sample can be determined.
DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is stained with a DNA-binding dye. For example, a PCR reaction producing a 400 base pair (bp) fragment would look like this on a gel:
Left lane: DNA ladder with 100, 200, 300, 400, 500 bp bands.
Right lane: result of PCR reaction, a band at 400 bp.
A DNA band contains many, many copies of the target DNA region, not just one or a few copies. Because DNA is microscopic, lots of copies of it must be present before we can see it by eye. This is a big part of why PCR is an important tool: it produces enough copies of a DNA sequence that we can see or manipulate that region of DNA.

Applications of PCR

Using PCR, a DNA sequence can be amplified millions or billions of times, producing enough DNA copies to be analyzed using other techniques. For instance, the DNA may be visualized by gel electrophoresis, sent for sequencing, or digested with restriction enzymes and cloned into a plasmid.
PCR is used in many research labs, and it also has practical applications in forensics, genetic testing, and diagnostics. For instance, PCR is used to amplify genes associated with genetic disorders from the DNA of patients (or from fetal DNA, in the case of prenatal testing). PCR can also be used to test for a bacterium or DNA virus in a patient's body: if the pathogen is present, it may be possible to amplify regions of its DNA from a blood or tissue sample.

Sample problem: PCR in forensics

Suppose that you are working in a forensics lab. You have just received a DNA sample from a hair left at a crime scene, along with DNA samples from three possible suspects. Your job is to examine a particular genetic marker and see whether any of the three suspects matches the hair DNA for this marker.
The marker comes in two alleles, or versions. One contains a single repeat (brown region below), while the other contains two copies of the repeat. In a PCR reaction with primers that flank the repeat region, the first allele produces a 200 start text, b, p, end text DNA fragment, while the second produces a 300 start text, b, p, end text DNA fragment:
Marker allele 1: primers flanking repeat region amplify a 200 bp fragment of DNA
Marker allele 2: primers flanking repeat region amplify a 300 bp fragment of DNA
You perform PCR on the four DNA samples and visualize the results by gel electrophoresis, as shown below:
The gel has five lanes:
First lane: DNA ladder with 100, 200, 300, 400, and 500 bp bands.
Second lane: DNA from crime scene, 200 bp band.
Third lane: Suspect #1 DNA, 300 bp band.
Fourth lane: Suspect #2 DNA, 200 and 300 bp bands.
Fifth lane: Suspect #3 DNA, 200 bp band.
Which suspect's DNA matches the DNA from the crime scene at this marker?
Choose 1 answer:

More about PCR and forensics

In real forensic tests of DNA from a crime scene, technicians would do an analysis conceptually similar to the one in the example above. However, a number of different markers (not just the single marker in the example) would be compared between the crime scene DNA and the suspects' DNA.
Also, the markers used in a typical forensic analysis don't come in just two different forms. Instead, they're highly polymorphic (poly = many, morph = form). That is, they come in many alleles that vary in tiny increments of length.
The most commonly used type of markers in forensics, called short tandem repeats (STRs), consist of many repeating copies of the same short nucleotide sequence (typically, 2 to 5 nucleotides long). One allele of an STR might have 20 repeats, while another might have 18, and another just 10start superscript, 1, end superscript.
By examining multiple markers, each of which comes in many allele forms, forensic scientists can build a unique genetic "fingerprint" from a DNA sample. In a typical STR analysis using 13 markers, the odds of a false positive (two people having the same DNA "fingerprint") are less than 1 in 10 start text, b, i, l, l, i, o, n, end textstart superscript, 1, end superscript!
Although we may think of DNA evidence being used to convict criminals, it has played a crucial role in exonerating falsely accused people (including some who had been jailed for many years). Forensic analysis is also used to establish paternity and to identify human remains from disaster scenes.

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