- Introduction to genetic engineering
- Intro to biotechnology
- DNA cloning and recombinant DNA
- Overview: DNA cloning
- Polymerase chain reaction (PCR)
- Polymerase chain reaction (PCR)
- Gel electrophoresis
- Gel electrophoresis
- DNA sequencing
- DNA sequencing
- Applications of DNA technologies
A technique used to amplify, or make many copies of, a specific target region of DNA.
- 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.
PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.
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 (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.
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 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.
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.
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:
- Denaturation (): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
- Annealing ( ): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
- Extension (): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
This cycle repeats times in a typical PCR reaction, which generally takes 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.
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 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
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 DNA fragment, while the second produces a 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.
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, to nucleotides long). One allele of an STR might have repeats, while another might have , and another just .
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 markers, the odds of a false positive (two people having the same DNA "fingerprint") are less than in !
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.
Want to join the conversation?
- Would you define "marker" a little better. I'm a little confused about it's meaning.(25 votes)
- It's a standardized test solution of specific marked DNA which allows scientists to have a comparison to the sample DNA placed in the wells.(20 votes)
- During the annealing process, isn't there a possibility of the DNA templates joining back to each other instead of the primer or what measures are taken to ensure that doesn't happen. Also, when does the polymerization of a cycle stops(6 votes)
- Taq polymerase is acquired from bacteria ,it is DNA pol III (responsible for elongation in prokaryotes) of a prokaryote. So, how can it elongate a eukaryotic DNA in PCR when it is meant to elongate a prokaryotic DNA?(8 votes)
- While there can be differences in the DNA from different species, those differences generally do not affect the ability to be copied by different DNA polymerases.
It is perhaps surprising, but we frequently take DNA from one organism and put it into another and have it replicated and even transcribed and translated!
(To do this we put the DNA into something called a vector, which provides the right signals to the host cell so that these processes can happen.)(3 votes)
- if we don't know the exact sequence of the gene, what ways can we can still use PCR to amplify that gene?(4 votes)
- PCR is usually used for amplification of known genes. Why? Because you need to have primers. how are you going to design or to use pre-existing primers if you do not know what sequence to they are aligned to?
So to start the PCR, one has to design degenerate primers.
First, to obtain reference sequence, use Bioinformatic software and databases NCBI, and Ensembl for finding most similar gene sequences from closely related species.
Once one finds it, use Ensembl to recreate exon sequences and therefore primers.
- What is a genetic marker?(2 votes)
- Genetic marker is known gene or DNA sequence (with a known location on the chromosome) used to identify individual or species.(6 votes)
- Are restriction enzymes used during PCR or are they the same as the primers?(2 votes)
- Hello dixit.anusha02,
At first - restriction enzymes are enzymes, i.e. biological macromolecules. Some of them occur naturally in cells to destroy damaged DNA.
Primers on the contrary are short artificial pieces of single-strand DNA, where the amplification starts. As you see, restriction enzymes and primers have quite little in common.
In the PCR you need the DNA sample, nucleotides, buffer solution and primers. The PCR is used to produce many identical DNA samples.
The restriction enzymes are used for a restriction digest, where the DNA is cut into pieces. This is useful for an analysis of the DNA (I can't explain it in a sentence). You often have to make a PCR before a restriction digest in order to have enough DNA for it, but as you see these are two different things. I hope this helps!(5 votes)
- What will happen if you add another primer between the two original primers? How many DNA strands will then be cloned?(2 votes)
- why does the primer stops after pairing with the required region of DNA instead of forming a complete copy of DNA ?(3 votes)
- A primer is just a couple nucleotides long, so it can't pair with the entire copy of DNA. It can only pair with the complementary nucleotides on the template strand.(1 vote)
- Why are multiple primers used when doing PCR?(2 votes)
- When scientists are cutting out the DNA fragment they want to copy, do they use restriction enzymes that produce sticky ends? And because they know the sticky end sequence, that is how they know the primer sequence? And so the primers would be complementary to each sticky end, and 'bind' to it through base pairing?
And if this is not correct, how do scientists know the primer sequence if they don't know the DNA sequence?
I'm not sure if this is correct, can someone please help out.(2 votes)
- You don't need to (and typically won't) cut the DNA before doing PCR.
If you use restriction enzymes (REs) then you usually already have enough DNA and can gel purify and use the cut fragment directly (e.g. ligate it into a vector cut with the same REs).
The recognition sequences for restriction enzymes are typically quite short (6 bp long is most usual). In contrast, primers are usually at least 18 nt long (often much longer) and so recognize a sequence that is on average at least 3 times longer. Thus, there isn't enough "information" (sequence) present from knowing a restriction site to design a PCR primer.
PCR is usually used to amplify small quantities of DNA into a large enough amount to use for something else (e.g. cloning into a vector).
This is typically done based on knowing the sequence you are trying to amplify.
If you don't know the exact sequence, but have some sequence information you can use "degenerate" primers, which are mixture of many similar (but non-identical) sequences.
(Examples where this can be used are when you have sequences from related organisms or amino acid but not nucleotide sequence.)
A technique that can be used if you need to amplify restriction fragments is to ligate short "adapter" sequences onto the ends of the restriction fragments. You can then amplify the sequences using primers that bind to the adapter sequences.
Does that help?(2 votes)