Restriction digest
Encyclopedia
A restriction digest is a procedure used in molecular biology
to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation (this term is used for other procedures as well). Hartl and Jones describe it this way:
The resulting digested DNA is very often selectively amplified using PCR, making it more suitable for analytical techniques such as agarose gel electrophoresis, and chromatography
. It is used in genetic fingerprinting
, and RFLP analysis
.
A given restriction enzyme
cuts DNA segments within a specific nucleotide sequence
, at what is called a restriction site.
These recognition sequence
s are typically four, six, eight, ten, or twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides which compose DNA (Adenine, Thymine, Guanine and Cytosine) into a four- to twelve-nucleotide sequence, recognition sequences tend to occur by chance in any long sequence. Restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories, and as a result, several potential "restriction sites" appear in almost any gene or locus of interest on any chromosome. Furthermore, almost all artificial plasmids include an (often entirely synthetic) polylinker
(also called "multiple cloning site") that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. This allows the insertion of almost any specific fragment of DNA into plasmid vectors
, which can be efficiently "cloned" by insertion into replicating bacterial cells.
After restriction digest, DNA can then be analysed using gel electrophoresis
. In gel electrophoresis, a sample of DNA is first "loaded" onto a slab of agarose gel (literally pipetted into small wells at one end of the slab). The gel is then subjected to an electric field, which draws the negatively charged DNA across it. The molecules travel at different rates (and therefore end up at different distances) depending on their net charge (more highly charged particles travel further), and size (smaller particles travel further). Since none of the four nucleotide bases carry any charge, net charge becomes insignificant and size is the main factor affecting rate of diffusion through the gel. Net charge in DNA is produced by the sugar-phosphate backbone
. This is in contrast to proteins, in which there is no "backbone", and net charge is generated by different combinations and numbers of charged amino acids
.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation (this term is used for other procedures as well). Hartl and Jones describe it this way:
This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present
.
The resulting digested DNA is very often selectively amplified using PCR, making it more suitable for analytical techniques such as agarose gel electrophoresis, and chromatography
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
. It is used in genetic fingerprinting
Genetic fingerprinting
DNA profiling is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier...
, and RFLP analysis
Restriction fragment length polymorphism
In molecular biology, restriction fragment length polymorphism, or RFLP , is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related...
.
A given restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
cuts DNA segments within a specific nucleotide sequence
DNA sequence
The sequence or primary structure of a nucleic acid is the composition of atoms that make up the nucleic acid and the chemical bonds that bond those atoms. Because nucleic acids, such as DNA and RNA, are unbranched polymers, this specification is equivalent to specifying the sequence of...
, at what is called a restriction site.
These recognition sequence
Recognition sequence
The recognition sequence, sometimes also referred to as recognition site, of any DNA-binding protein motif that exhibits binding specificity, refers to the DNA sequence , to which the domain is specific...
s are typically four, six, eight, ten, or twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides which compose DNA (Adenine, Thymine, Guanine and Cytosine) into a four- to twelve-nucleotide sequence, recognition sequences tend to occur by chance in any long sequence. Restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories, and as a result, several potential "restriction sites" appear in almost any gene or locus of interest on any chromosome. Furthermore, almost all artificial plasmids include an (often entirely synthetic) polylinker
Multiple cloning site
A multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...
(also called "multiple cloning site") that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. This allows the insertion of almost any specific fragment of DNA into plasmid vectors
Vector (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
, which can be efficiently "cloned" by insertion into replicating bacterial cells.
After restriction digest, DNA can then be analysed using gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
. In gel electrophoresis, a sample of DNA is first "loaded" onto a slab of agarose gel (literally pipetted into small wells at one end of the slab). The gel is then subjected to an electric field, which draws the negatively charged DNA across it. The molecules travel at different rates (and therefore end up at different distances) depending on their net charge (more highly charged particles travel further), and size (smaller particles travel further). Since none of the four nucleotide bases carry any charge, net charge becomes insignificant and size is the main factor affecting rate of diffusion through the gel. Net charge in DNA is produced by the sugar-phosphate backbone
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. This is in contrast to proteins, in which there is no "backbone", and net charge is generated by different combinations and numbers of charged amino acids
Proteinogenic amino acid
Proteinogenic amino acids are those amino acids that can be found in proteins and require cellular machinery coded for in the genetic code of any organism for their isolated production. There are 22 standard amino acids, but only 21 are found in eukaryotes. Of the 22, 20 are directly encoded by...
.
Possible Uses
Restriction digests are necessary for performing any of the following analytical techniques:- RFLP - Restriction Fragment Length Polymorphism
- AFLP - Amplified Fragment Length Polymorphism
- STRP - Short Tandem Repeat Polymorphism
Various restriction enzymes
There are numerous types of restriction enzymes, each of which will cut DNA differently. (See article on Restriction enzymes for examples). There are some that cut a three base pair sequence while others can cut four, six, and even eight. Each enzyme has distinct properties that determine how efficiently it can cut and under what conditions. Most manufacturers that produce such enzymes will often provide a specific buffer solution that contains the unique mix of cations and other components that aid the enzyme in cutting as efficiently as possible. Different restriction enzymes also have different optimal temperatures under which they function.See also
- Agarose gel electrophoresis
- DNA sequencingDNA sequencingDNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
- FERMENTAS
- Genetic fingerprintingGenetic fingerprintingDNA profiling is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier...
- PCR
- Restriction fragment length polymorphismRestriction fragment length polymorphismIn molecular biology, restriction fragment length polymorphism, or RFLP , is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related...
External links
- New England Biolabs - Producer of restriction enzymes. This site contains highly detailed information on numerous enzymes, their optimal temperatures, and recognition sequences.
- REBASE