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    Restriction enzymes & DNA ligase (article)

    Restriction digestion. Sticky ends and blunt ends. Ligation reactions.

    Key points:

    Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one or a few target sequences and cuts DNA at or near those sequences.

    Many restriction enzymes make staggered cuts, producing ends with single-stranded DNA overhangs. However, some produce blunt ends.

    DNA ligase is a DNA-joining enzyme. If two pieces of DNA have matching ends, ligase can link them to form a single, unbroken molecule of DNA.

    In DNA cloning, restriction enzymes and DNA ligase are used to insert genes and other pieces of DNA into plasmids.

    How do you cut and paste DNA?

    In DNA cloning, researchers make many copies of a piece of DNA, such as a gene. In many cases, cloning involves inserting the gene into a piece of circular DNA called a plasmid, which can be copied in bacteria.

    How can pieces of DNA from different sources (such as a human gene and a bacterial plasmid) be joined together to make a single DNA molecule? One common method is based on restriction enzymes and DNA ligase.

    A restriction enzyme is a DNA-cutting enzyme that recognizes specific sites in DNA. Many restriction enzymes make staggered cuts at or near their recognition sites, producing ends with a single-stranded overhang.

    If two DNA molecules have matching ends, they can be joined by the enzyme DNA ligase. DNA ligase seals the gap between the molecules, forming a single piece of DNA.

    Restriction enzymes and DNA ligase are often used to insert genes and other pieces of DNA into plasmids during DNA cloning.

    Restriction enzymes

    Restriction enzymes are found in bacteria (and other prokaryotes). They recognize and bind to specific sequences of DNA, called restriction sites. Each restriction enzyme recognizes just one or a few restriction sites. When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule. Typically, the cut is at or near the restriction site and occurs in a tidy, predictable pattern. [Why do bacteria have restriction enzymes?]

    As an example of how a restriction enzyme recognizes and cuts at a DNA sequence, let's consider EcoRI, a common restriction enzyme used in labs. EcoRI cuts at the following site:

    5'-...GAATTC...-3' 3'-...CTTAAG...-5'

    EcoRI site

    When EcoRI recognizes and cuts this site, it always does so in a very specific pattern that produces ends with single-stranded DNA “overhangs”:

    An EcoRI enzyme binds to an EcoRI site in a piece of DNA and makes a cut on both strands of the DNA. The pattern of the cut is:

    5'-...G|AATTC...-3' 3'-...CTTAA|G...-5'

    Thus, it produces an overhang of 5'-AATT-3' on each end of the cut DNA.

    If another piece of DNA has matching overhangs (for instance, because it has also been cut by EcoRI), the overhangs can stick together by complementary base pairing. For this reason, enzymes that leave single-stranded overhangs are said to produce sticky ends. Sticky ends are helpful in cloning because they hold two pieces of DNA together so they can be linked by DNA ligase.

    Not all restriction enzymes produce sticky ends. Some are “blunt cutters,” which cut straight down the middle of a target sequence and leave no overhang. The restriction enzyme SmaI is an example of a blunt cutter:

    A SmaI enzyme binds to the SmaI restriction site, which is:

    5'-...CCCGGG...-3' 3'-...GGGCCC...5'

    It makes a cut right in the middle of this sequence on both strands, producing blunt ends. The cut sites are:

    5'-...CCC|GGG...-3' 3'-...GGG|CCC...5'

    Blunt-ended fragments can be joined to each other by DNA ligase. However, blunt-ended fragments are harder to ligate together (the ligation reaction is less efficient and more likely to fail) because there are no single-stranded overhangs to hold the DNA molecules in position.

    [Where do restriction enzymes get these weird names?]

    DNA ligase

    If you’ve learned about DNA replication, you may already have met DNA ligase. In DNA replication, ligase’s job is to join together fragments of newly synthesized DNA to form a seamless strand. The ligases used in DNA cloning do basically the same thing. If two pieces of DNA have matching ends, DNA ligase can join them together to make an unbroken molecule.

    Fragment 1 of DNA: 5'-...G 3'-...CTTAA Fragment 2 of DNA: AATTC...-3' G...-5'

    The single-stranded regions of the two molecules can stick together by hydrogen bonding, but there are still gaps in the backbone:

    5'-...G|AATTC...-3' 3'-...CTTAA|G...-5'

    DNA ligase seals the gaps to make an unbroken molecule of DNA:

    5'-...GAATTC...-3' 3'-...CTTAAG...-5'

    How does DNA ligase do this? Using ATP as an energy source, ligase catalyzes a reaction in which the phosphate group sticking off the 5’ end of one DNA strand is linked to the hydroxyl group sticking off the 3’ end of the other. This reaction produces an intact sugar-phosphate backbone.

    Source : www.khanacademy.org

    LS 7A wk 8 Questions Flashcards

    Start studying LS 7A wk 8 Questions. Learn vocabulary, terms, and more with flashcards, games, and other study tools.

    LS 7A wk 8 Questions

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    Which of the following BEST describes the way you would engineer bacterial cells to produce a human protein?

    Click card to see definition 👆

    Use restriction enzymes to cleave both the donor DNA and the vector DNA.

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    What features of DNA make it possible to make recombinant DNA in the lab? (Select all that apply.)

    Click card to see definition 👆

    1. The genetic code is the same for all organisms.

    2. Restriction enzymes cut DNA from all species.

    3. Two double helices from different sources can be ligated together.

    Click again to see term 👆

    1/36 Created by porkilton8

    Terms in this set (36)

    Which of the following BEST describes the way you would engineer bacterial cells to produce a human protein?

    Use restriction enzymes to cleave both the donor DNA and the vector DNA.

    What features of DNA make it possible to make recombinant DNA in the lab? (Select all that apply.)

    1. The genetic code is the same for all organisms.

    2. Restriction enzymes cut DNA from all species.

    3. Two double helices from different sources can be ligated together.

    A graduate student wants to create a recombinant DNA molecule and introduce this molecule into bacteria. What is the CORRECT order of steps that he should follow?

    choose plasmid/donor DNA → cut with restriction enzymes → join fragments via DNA ligase → transform bacteria

    The transformation step in creating bacteria genetically engineered to produce human proteins involves:

    bacteria taking up the recombinant DNA in the form of the vectors.

    One characteristic of restriction enzymes is that they cut:

    double-stranded DNA strands at specific sites.

    If a restriction site of AatII is 5'-GACGTC -3' then 3'-GACGTC-5' is also an AatII restriction site.

    False

    What is the name of the class of enzymes that recognizes and cuts a specific sequence of DNA?

    restriction enzymes

    Restriction enzymes recognize certain DNA sequences and:

    some of them will cut straight through, while others will leave an overhang at both ends of the cut.

    A Southern blot is a technique that relies on hybridization of:

    a nucleic acid probe to a complementary DNA.

    A DNA molecule is cut with two different restriction enzymes known to cleave it only once each. After gel electrophoresis, three different DNA fragments are detected. This result means that the original DNA molecule was:

    linear.

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    restriction enzyme

    restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensible tools of recombinant DNA technology (genetic engineering). A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so

    restriction enzyme

    biology

    Alternate titles: restriction endonuclease

    By The Editors of Encyclopaedia Britannica • Edit History

    cDNA library See all media

    Key People: Hamilton O. Smith Werner Arber

    Related Topics: nuclease type IV restriction enzyme type I restriction enzyme type III restriction enzyme type II restriction enzyme

    See all related content →

    restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensible tools of recombinant DNA technology (genetic engineering).

    A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.

    Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions are called recognition sequences, or recognition sites, and are randomly distributed throughout the DNA. Different bacterial species make restriction enzymes that recognize different nucleotide sequences.

    When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides. Bacteria prevent their own DNA from being degraded in this manner by disguising their recognition sequences. Enzymes called methylases add methyl groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

    Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors. Types I and III enzymes are similar in that both restriction and methylase activities are carried out by one large enzyme complex, in contrast to the type II system, in which the restriction enzyme is independent of its methylase. Type II restriction enzymes also differ from types I and III in that they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly, sometimes hundreds of bases from the recognition sequence. Several thousand type II restriction enzymes have been identified from a variety of bacterial species. These enzymes recognize a few hundred distinct sequences, generally four to eight bases in length. Type IV restriction enzymes cleave only methylated DNA and show weak sequence specificity.

    Restriction enzymes were discovered and characterized in the late 1960s and early 1970s by molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. The ability of the enzymes to cut DNA at precise locations enabled researchers to isolate gene-containing fragments and recombine them with other molecules of DNA—i.e., to clone genes. The names of restriction enzymes are derived from the genus, species, and strain designations of the bacteria that produce them; for example, the enzyme EcoRI is produced by Escherichia coli strain RY13. It is thought that restriction enzymes originated from a common ancestral protein and evolved to recognize specific sequences through processes such as genetic recombination and gene amplification.

    The Editors of Encyclopaedia BritannicaThis article was most recently revised and updated by Kara Rogers.

    Source : www.britannica.com

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