DNA amplification

Annealing, cloning, denaturation, DNA, DNA amplification, DNA cloning, DNA fingerprinting, DNA replication, elongation, extension, molecular biology, paternity testing, PCR, polymerase, real-time PCR.

DNA, short for deoxyribonucleic acid, is the name of the complex biological compound that makes up the genetic "blueprint" to make a living organism. The unique twisted ladder shape of DNA is called a double helix. The sides of DNA's double helix are made of alternating sugar and phosphate molecules while the "rungs" of the "ladder" are made of corresponding pairs of small molecules (each one half of the "rung") called bases. There are four different types of bases in DNA: adenine (A), thymine (T), cytosine (C), and guanine (G); the order in which they occur is referred to as the DNA sequence. Each base has a complement; that is, a base it usually pairs with: adenine is complementary to thymine, and cytosine to guanine. Due to their chemical structure these molecules are sometimes called nitrogenous or nucleotide bases. A sequence of three base pairs is called a codon. A codon is the fundamental unit of the genetic code, with each of the 64 possible 3-base sequences corresponding to a particular instruction: start, stop, or one of 20 amino acids. The biochemical machinery within a cell "reads" a DNA strand, eventually translating a series of codons into long chains of amino acids. These amino acid-chains fold to form proteins, complex molecules whose structure and function underlie all biological life. In all, the human genome, one complete copy of all the DNA containing the code for a human being, is three billion base pairs long. However, only a small percentage of the genome codes for proteins or has any other known function.

Polymerase chain reaction (PCR) is a scientific method used commonly in molecular biology. Its name is based on one of its key components, an enzyme (biomolecules which aid certain chemical reactions) called DNA polymerase, which is used to replicate a piece of DNA. During PCR, each strand of DNA's double helix is separated (that is, the "ladder" of DNA is divided lengthwise, as if cut through the "rungs") from the other with heat (a process called denaturation) and then used by DNA polymerase as a template to make the complementary strand. Each replicated piece of DNA can then serve as a template for further replication, setting in motion a chain reaction in which the selected sequence of DNA is exponentially replicated. Two strands become four, four become eight, and so on. The amplified DNA product (so called as it has been replicated many times over) can then be used for a variety of molecular biological tests and procedures, often by allowing detection or large scale production of a particular sequence. One example is DNA cloning, wherein a DNA sequence is isolated and amplified in a living organism. Another is using PCR to allow detection of a genetic mutation in an individual suspected of having a genetic disorder.

The PCR technique was developed in 1983 and is generally credited to Kary Mullis; he later won the Nobel Prize for this work. PCR is now commonly used in medical and biological research in a variety of applications, including DNA cloning, functional analysis of genes, diagnosis of hereditary diseases, genetic fingerprinting for use in forensics and paternity testing, and the detection and diagnosis of infectious diseases.

General: Polymerase chain reaction (PCR) is a scientific method commonly used in molecular biology (the study of the complex chemical components such as proteins and DNA which underpin life). Its name is based on one of its key components, DNA polymerase, an enzyme (a biomolecule which aids certain chemical reactions) which is used to replicate DNA. During PCR, each strand of DNA's double helix is separated from the other with heat (a process called denaturation) and then used by DNA polymerase as a template to make the complementary strand (DNA polymerase working on a template strand ATCG would produce a strand made up of a sequence of the corresponding base pairs TAGC). The reaction is conducted in a medium where the key components for making new DNA are readily available such that each newly created strand of DNA can then serve as a template for further copying, setting in motion a chain reaction in which the selected sequence of DNA is exponentially replicated. DNA polymerases always copy in the same direction along a particular strand and always in opposing directions along two complementary strands. In this way two complementary strands become four, four become eight, and so on. PCR usually consists of a series of 20 to 40 repeated temperature cycles, with each cycle typically consisting of two to three discrete temperature steps. This temperature cycling enables PCR by successively denaturing the initial DNA sample and then allowing DNA primers (short complementary strands of DNA that serve as the attachment point for DNA polymerase) to pair with the newly freed strands, and DNA polymerase to attach and replicate. The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90?C), and followed by one at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of divalent ions and deoxynucleoside triphosphates (dNTPs), which are the building blocks for making a new DNA strand, and the melting temperature of the primers.

Initiation: This step is not performed for every PCR. Initiation consists of heating the reaction to a temperature of 94-96?C for one to nine minutes, and is necessary only for DNA polymerases that require heat activation by hot-start PCR. During hot-start PCR, the reaction components are "preheated" before the addition of the polymerase. Hot-start PCR reduces the amplification of nonspecific products during the initial setup stages of PCR so that more specific products are amplified during the actual PCR.

Denaturation: Denaturation is the heat-induced separation of the DNA double strands into single strands (that is, the "ladder" of DNA is divided lengthwise, as if cut through the "rungs"). This step is the first regular cycling event and consists of heating the reaction to 94-98?C for 20-30 seconds. It causes melting of the DNA template and primers by disrupting the hydrogen bonds between the complementary bases of the DNA strands, yielding single strands of DNA.

Annealing: During the annealing step, the reaction temperature is lowered to 50-65?C for 20-40 seconds to allow for annealing (bonding) of the primers to the single-stranded DNA template. Typically, the annealing temperature is about 3-5 ?C below the melting temperature of the primers used. Stable DNA-DNA hydrogen bonds are formed only when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. Almost all PCR applications use a heat-stable DNA polymerase such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. DNA polymerase assembles a new DNA strand from nucleotides, base by complementary base, by using the single-stranded DNA as a template. DNA primers are synthesized such that they attach to opposing ends (the "head" or "foot" of the ladder) of the two, heat-separated complementary strands that comprise the sequence targeted for amplification. As DNA polymerase may only attach to primed locations, this guarantees that only the desired section of DNA will be replicated (each strand composing one half of the DNA "ladder" defining that section).

Extension/elongation: During the extension/elongation step, the temperature applied depends on the DNA polymerase used. Taq polymerase, which is frequently used because it is stable in high temperatures, works best at 75-80?C. At this step, the DNA polymerase synthesizes a new, complementary DNA strand by adding the corresponding dNTPs to the template (in essence "repairing" the "ladder" that had been previously split). The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified, though a general guide value is 1,000 bases added per minute.

Reagents: Several reagents are needed for PCR. These include a DNA template that contains the sequences to be amplified; primers complementary to the two opposing ends of each of the strands of the region of DNA being amplified; DNA polymerase; deoxynucleoside triphosphates (nucleotide bases); buffer solution, which provides a suitable environment for the reaction; and magnesium, manganese, and potassium ions, which are needed for the DNA polymerase to work.

Final elongation: A final elongation step is added on occasion to ensure that any remaining single-strands of DNA are fully extended.

Several variations of polymerase chain reaction (PCR) are commonly used in medical and biological research in a variety of applications, including DNA cloning, functional analysis of genes, diagnosis of hereditary diseases, genetic fingerprinting for use in forensics and paternity testing, and the detection and diagnosis of infectious diseases. Reverse-transcriptase PCR (RT-PCR) uses a polymerase from a retrovirus (e.g. HIV), which transcribes the DNA complement of a strand of RNA (ribonucleic acid), a molecule similar to DNA. This variant of PCR can be used to detect which genes (a sequence of DNA which defines a particular trait) are being expressed, that is, which portions of DNA are actively being decoded to enact some cellular function, the first step of which is transcribing a strand of RNA from a DNA template. Real-time PCR is currently considered the most sensitive, specific, and rapid form of PCR. During real-time PCR, the PCR product is quantified while the process is occurring, allowing the total amount of replicated DNA to be determined after each round of amplification. This method is often combined with RT-PCR to measure the level of gene expression. This is accomplished by comparing the time it took to detect the amplified product (often by means of a fluorescent dye) to previously determined standards. Another commonly used form of PCR is allele-specific PCR which is used to identify genetic variations known as single-nucleotide polymorphisms (SNPs). Alleles are variants of a single gene and SNPs are mutations in which a gene sequence differs by only one base. Alleles are often differentiated by only an SNP. SNPs have been found to be the cause of many human diseases and are, therefore, of increasing interest to medical researchers.

DNA cloning: DNA cloning is the process of isolating a defined DNA sequence and obtaining multiple copies of it in a living organism. Cloning is frequently used to amplify DNA fragments containing genes. PCR allows for the production of large quantities of DNA that can be readily cloned and used to study the functions and behavior of genes in living systems. DNA cloning involves four basic steps. First, the source and vector DNA fragments (strands of DNA used to insert a desired sequence into the organism) are isolated and freed from contaminants. Then restriction enzymes, molecules that cut DNA at particular locations, are used to slice up these two strands to create ends that can connect the source DNA with the vector. Next, the source's DNA is bonded to the vector's with a DNA ligase, an enzyme that repairs the cuts and creates a single length of DNA. Finally the DNA is transformed into a host cell, generally a bacterium or other easily cultured organism. PCR-mediated cloning is a family of methods rather than a single technique. TA cloning, for example, uses Taq polymerase or one of a group of other polymerases that preferentially add the base adenine to particular ends of PCR products.

Functional analysis of genes: For many organisms, genes are encoded in long strands of DNA. Genes encode for proteins that are made when the gene is expressed. When a gene is expressed, the DNA that contains that gene is translated into a piece of RNA, a molecule similar to DNA in that it has long chains of nucleic acid bases, but different in that it is usually single stranded. PCR can be used to amplify genes of interest to researchers so their function can be studied.

Diagnosis of hereditary genes: A genetic disorder is a disease or condition caused by a defect in an individual's genes. In human beings, each cell contains a full copy of the genetic code. This code is arranged in 23 pairs of structures called chromosomes. Genes are passed down among family members, and individuals receive two copies of most genes, one on each chromosome received from either parent. When inherited, genetic disorders can follow an autosomal dominant pattern of inheritance, meaning that only one copy of the defective gene is necessary for the condition to appear. They can also follow an autosomal recessive pattern of inheritance, meaning that two copies of the defective gene, one from each parent, must be inherited for the condition to appear. Disorders which can only be passed down from the mother to the child or from a father to a son, are called X or Y-linked traits, respectively, as the genes involved appear on the X or Y chromosomes (the chromosomes responsible for determining the sex of an individual). Females inherit two X chromosomes, one from her mother and one from her father, whereas a male inherits an X chromosome from his mother and a Y chromosome from his father.

One particular hurdle in studying genes involved in genetic disorders is that samples often contain only very small amounts of DNA and sequences of interest are difficult to separate or detect apart from the remainder of the sample. PCR can be used to amplify the DNA that contains a defective gene in order to determine whether an individual has a particular genetic disease.

Identification of genetic fingerprints for use in forensics: Forensics is the application of science to answer questions of interest to the criminal justice system. DNA fingerprinting is used by forensic scientists to assist in the identification of individuals based on their unique genetic makeup. PCR is used to take the small amount of DNA present at a crime scene, for instance, and increase the amount available for study.

Identification of genetic fingerprints for use in familial determination: PCR can be used to help determine whether and how closely two persons are related. Comparing the DNA sequence of two individuals can demonstrate whether one was derived from the other or if the two had similar parentage. During a paternity test, a cotton swab is used to scrape the inside of the cheeks of the potential relatives. Since so little genetic information is obtained from that one sample, PCR is used to increase the available amount so that the two samples can be compared to determine whether the genetic information is the same.

Detection and diagnosis of infectious disease: PCR is often employed in the diagnosis of various bacterial and viral infections, such as the human immunodeficiency virus (HIV) and tuberculosis. PCR is used to amplify samples of the infectious agent for identification.

DNA sequencing: DNA makes up the genetic "blueprint" that contains all the information necessary to make a living organism. The unique twisted ladder shape of DNA is called a double helix. The sides of the double helix are made of alternating sugar and phosphate molecules. DNA sequencing is the process of determining the order of nucleotide bases (small molecules that comprise the "rungs" of the "ladder") that appear in a particular strand of DNA. Four different types of bases occur in DNA: adenine (A), thymine (T), cytosine (C), and guanine (G).

The Human Genome Project is an international scientific research project whose primary goal is to determine the sequence of base pairs that make up human genetic code, and to identify the approximately 20,000-25,000 genes that make up a human being. Once the human genome is mapped out, it will be possible for researchers to compare an individual's DNA to this template to determine whether that person has a particular genetic mutation that may cause a disease. Although this information is useful, it is also a cause for ethical concern, as this information could be used discriminatorily, such as in the denial of health insurance to those with identified genetic risk factors.

Polymerase chain reaction (PCR), particularly real-time PCR, has changed the way microbiology laboratories diagnose many human infections. For example, the technique has improved the detection of mycobacterial infections, and allowed for the rapid differentiation of mycobacterial species, quantification of load, and detection of drug resistance in these organisms.

Legal implications: The same kinds of data that are used to analyze genetic differences between humans for medical purposes are also used in courts of law to determine identity. DNA fingerprinting is used by forensic scientists to assist in the identification of individuals based on their unique genetic code. (Forensics is the application of science to answer questions of interest to the criminal justice system.) PCR is used to take the small amount of DNA present at a crime scene, for instance, and increase the amount available for study. It can also be used in questions of inheritance or paternity disputes.

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As genetic research and technique advances, a number of new applications based on the fine manipulation of genetic material will likely become an increasingly important part of modern healthcare. As many of these new techniques will likely rely on the mass production of particular sequences of DNA, PCR will likely play a critical role.

The Human Genome Project is an international scientific research project whose primary goal is to determine the sequence of base pairs that make up human genetic code, and to identify the approximately 20,000-25,000 genes that make up a human being. Once the human genome is mapped out, it will be possible for researchers to compare an individual's DNA to this template to determine whether that person has a particular genetic mutation that may cause a disease. Related projects which map out the human proteome, or the entire complement of proteins that the genes of the human genome code for, are also currently underway, much of which rely, as the continuing genomic work, upon PCR.

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

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