AFLP

Related Terms

Amplified fragment length polymorphism analysis, AFLP, DNA fingerprinting, genetic diversity, PCR, polymerase chain reaction, single nucleotide polymorphism, SNP.

Background

The molecule deoxyribonucleic acid (DNA), found in the nucleus of every cell of every organism, contains the blueprints for that organism's growth and function. A gene is a short segment of DNA that can be assigned a function, such as designating the structure of a protein. All of the genes in an organism are collectively called the genome. Many segments of DNA do not code for proteins. Some turn genes on and some turn them off, but most genes are poorly understood at this time.
DNA is a long, complex chemical represented by a code of four nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). Consequently, a code may read: "AATGCGCCTTTGAGGTC," and so on. Three letters in sequence designate an amino acid. Amino acids arranged linearly form a protein. Proteins are organic compounds required for the growth and maintenance of the body. Some proteins make nonprotein chemicals, such as cortisone and adrenaline, which the body needs to function.
The DNA of all humans is 99.9% identical. The remaining 0.1% accounts for all of the differences observed among individuals. A single nucleotide polymorphism (SNP, pronounced "snip") is a natural variation in the population. SNPs account for at least 90% of that 0.1%.
Polymerase chain reaction (PCR) is a molecular biology technique used to make copies of DNA for research. Normally, DNA is present in amounts too small for study. PCR uses special enzymes to make millions of copies of a specific piece of DNA. As PCR progresses, the DNA generated is used as a template for replication. This sets in motion a chain reaction in which the DNA template is amplified. With PCR it is possible to amplify a one or a few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the piece of DNA. PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene or part of a gene.
Amplified fragment length polymorphism-polymerase chain reaction (AFLP-PCR), commonly called "DNA fingerprinting," is a highly sensitive and reproducible method for detecting SNPs in DNA. AFLP technology can detect various polymorphisms in different areas of the genome simultaneously. Forensic scientists may use DNA fingerprinting to identify individuals based on their DNA profiles. AFLP can also be used to identify progenitors, siblings, or paternity. Comparing the DNA sequence of an individual to that of another individual can determine whether one of them was derived from the other. Specific sequences are usually examined to see whether they were copied verbatim from one individual's genome to the other. If that was the case, then this proves that the genetic material of one individual could have been derived from that of the other (i.e., one is the parent of the other). It can also be used to assess the genetic diversity of a population of cells, for example, in determining the homogeneity of tumor cells. This technique is used to determine whether the cells that make up a tumor have the same DNA mutation or if they have varying mutations.
The widespread use of the DNA fingerprinting technique has revolutionized forensic science and has been used to resolve paternity disputes. Modern AFLP-PCR technology was developed by KeyGene, a private cooperation that conducts genetic and genomic research. Their technology has become one of the most popular genetic fingerprinting technologies used throughout the world.

Methods

Amplified fragment length polymorphism (AFLP), also known as DNA fingerprinting, is a highly sensitive and reproducible method for detecting single nucleotide changes, or polymorphisms, in DNA. This technique detects multiple DNA fragments by means of polymerase chain reaction (PCR) amplification. PCR is a molecular biology technique used to make copies of DNA for research.
Normally, DNA is present in amounts too small for study. PCR uses special enzymes to make millions of copies of a specific piece of DNA. As PCR progresses, the DNA generated is used as a template for replication. This sets in motion a chain reaction in which the DNA template is amplified. With PCR it is possible to amplify one or a few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the piece of DNA. PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene or part of a gene.
AFLP technology is performed in several steps. First, genomic DNA samples are cut at specific sites by two restriction enzymes. This cutting is followed by the ligation, or joining, of double-stranded oligonucleotide adapters, which are short, chemically synthesized, double-stranded DNA molecules used to link the ends of two other DNA molecules.
Next, PCR is used to amplify some of these fragments. Fragments are then separated for identification using electrophoresis. Electrophoresis is another molecular biology technique that uses an electrical current and a gel membrane to separate DNA fragments based on their size. The gel membranes used for electrophoresis differ in their composition and the size of the pores in the membrane. Certain gel membranes will be used based on the known size of the DNA fragment that is being studied. The other aspect of gel electrophoresis includes an electric current used to move the fragments through the gel; the speed at which they move through the gel determines their size. After the DNA fragments are separated on the gel membrane, the gel can be stained so the DNA fragments, which show as small bands on the gel, can be visualized.
A typical AFLP fingerprint pattern consists of 50-100 amplified fragments of various sizes on a gel. When comparing two or more samples, the frequency of matching AFLP markers, meaning the presence or absence of a particular pattern in the DNA sequence, depends on the level of single nucleotide polymorphisms (SNPs) between the tested DNA samples. AFLP polymorphisms are typically SNPs in the DNA restriction sites, which are specific nucleotide sequences. Genomic deletions, insertions, and rearrangements affecting the size or presence of restriction fragments also produce detectable polymorphisms.
AFLP technology can be used with a wide variety of restriction enzymes and all feasible combinations of selective nucleotides. Individual samples can be fingerprinted using more or different enzyme and primer combinations. The application of the AFLP technology is not dependent on the cataloged DNA sequence database.

Research

AFLP-PCR is used in linked marker assay development, where a linked marker is a specific fragment from a fingerprint that is present only when a specific trait is present in the organism. This fragment represents a DNA region near the gene region coding for this trait and allows investigators to generate maps for quantitative trait locus (QTL) analysis.

Implications

DNA fingerprinting using amplified fragment length polymorphism (AFLP) technology is increasingly used in many technologies, including legal and forensic applications. DNA fingerprinting is used by forensic scientists to help identify individuals based on their DNA profiles. AFLP can also be used to identify progenitors, siblings, or paternity. Comparing the DNA sequence of an individual to that of another can show whether one of them was derived from the other. Specific sequences are usually looked at to see whether they were copied verbatim from one individual's genome to the other. If that was the case, then this proves that the genetic material of one individual could have been derived from that of the other (i.e., one is the parent of the other). It can also be used to assess the genetic diversity of a population of cells, for example, in determining the homogeneity of tumor cells. This technique is used to determine whether the cells that make up a tumor have the same DNA mutation or varying mutations.
AFLP-PCR is also used to detect food-borne illnesses in the food chain, which may lead to the production of healthier foods. Genetically modified animals are increasingly becoming important model systems for studying diseases and testing new pharmaceuticals.
This technology has also revolutionized the study of ancestry by providing a molecular DNA map, which is a characteristic blueprint of genealogies, or family histories, and the genomic differences between ethnic groups in humans.

Limitations

Nonidentical comigrating bands in the amplified fragment length polymorphism (AFLP) fingerprints may contribute noise instead of signal to the dataset without being detected.
As with any methodology that uses polymerase chain reaction (PCR), extreme caution and proper control samples must be used to ensure that there is no cross-contamination of samples during manipulation. The most common cause of cross-contamination is human error and can be something as simple as not changing the tip of a pipette, an instrument used to add or remove solutions for a sample in molecular biology.

Future research

Amplified fragment length polymorphism (AFLP) technology will continue to be used to map the genetic diversity of human, animal, plant, and microbial populations throughout the world. AFLP is increasingly automated, allowing for mapping and database storage of more and more samples. With continued mapping and study of the human genome, scientists will likely identify DNA regions associated with diseases and traits. In the future, single nucleotide polymorphism (SNPs) may offer information about predisposition to certain diseases through identification of the changes in DNA.
Such information has broad ethical implications for fair use and genetic privacy. On May 21, 2008, President George W. Bush signed into law the Genetic Information Nondiscrimination Act (GINA), which prohibits U.S. insurance companies and employers from discriminating on the basis of information derived from genetic tests. This act forbids insurance companies from discriminating through reduced coverage or pricing and prohibits employers from making adverse employment decisions based on an individual's genetic code. In addition, insurers and employers are not allowed under the law to request or demand a genetic test.

Author information

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

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