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How does PCR work?
What is PCR amplification?
How does PCR amplify DNA?
Which enzyme is used for PCR amplification?
Aug 17, 2020 · To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes - builds - two new strands of DNA, using the original strands as templates.
- Overview
- Key points:
- What is PCR?
- Taq polymerase
- PCR primers
- The steps of PCR
- Using gel electrophoresis to visualize the results of PCR
- Applications of PCR
- Sample problem: PCR in forensics
- More about PCR and forensics
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.
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.
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°C (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 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.
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.
[More detailed diagram showing DNA and primer directionality]
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°C ): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
2.Annealing (55 - 65 °C ): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
3.Extension (72°C ): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
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.
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:
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.
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 bp DNA fragment, while the second produces a 300 bp DNA fragment:
You perform PCR on the four DNA samples and visualize the results by gel electrophoresis, as shown below:
Which suspect's DNA matches the DNA from the crime scene at this marker?
Choose 1 answer:
Choose 1 answer:
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 10 1 .
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 billion 1 !
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.
[Attribution and references]
Mar 6, 2023 · The polymerase chain reaction (PCR) is a laboratory nucleic acid amplification technique used to denature and renature short segments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences using DNA polymerase I enzyme, an isolate from Thermus aquaticus, known as Taq DNA.
- 2023/03/06
The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours.
The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study.
4 days ago · The polymerase chain reaction enables investigators to obtain the large quantities of DNA that are required for various experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics.
Dec 24, 2022 · PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.
