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    describe the role of restriction enzymes and dna ligase in the process of making recombinant plasmids during the process of plasmid dna transformation.

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    What is the role of restriction enzymes, DNA ligase, and cloning vectors in generating recombinant DNA?

    Answer to: What is the role of restriction enzymes, DNA ligase, and cloning vectors in generating recombinant DNA?

    Biotechnology

    What is the role of restriction enzymes, DNA ligase, and cloning vectors in generating...

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    Question:

    What is the role of restriction enzymes, DNA ligase, and cloning vectors in generating recombinant DNA?

    Recombination:

    The process involving the production of variations in the gene combinations by combining pieces of DNA is known as recombination. The pieces are created by breaking the strands from the original sequence. This process also occurs naturally in the cell during the creation of gametes.

    Answer and Explanation:

    Restriction enzymes: This enzyme is responsible for creating cuts in the sequence of DNA. The nucleotides located in a particular site will be identified by the enzyme where the process will occur. This restriction site will be present in the DNA molecule of the cell and the DNA molecule that has been introduced in the cell for creating recombinant.DNA ligase enzyme: This enzyme is responsible for combining the fragments of DNA that have been created after the cuts. The foreign piece will be added to the DNA molecule of the cell by the action of the enzyme.Cloning Vector: These vectors are introduced in the cell containing the piece of DNA that will be added to the cell's genetic material. The vector will have the particular restriction sites at the ends of the target DNA, allowing its release.

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    How Ligase is Used to Engineer Recombinant DNA

    from

    Chapter 54 / Lesson 4

    12K

    In relation to recombinant DNA engineering, DNA ligase conjoins two DNA molecules while restriction enzymes cut DNA molecules. Explore how DNA ligase and restriction enzymes work together to facilitate recombinant DNA technology.

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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

    Step Two: Make Recombinant Plasmids

    Step Two: Make Recombinant Plasmids

    If DNA ligase was added to the tube of cut DNA, the sugar-phosphate backbone bonds would be sealed and the two cut linear DNA molecules would become one circular recombinant plasmid. This plasmid will have an unaltered Amp antibiotic resistance gene and an inactivated LacZ gene with the insertion. The inactivation of one gene on the plasmid while leaving the other gene unchanged is a key requirement for gene cloning.

    Fig. 9: Foreign DNA with gene of interest (A, black) and plasmid (B) cut with same restriction enzyme. (Image by D. Lee)

    Fig. 10: Cut foreign DNA and plasmid mixed (A), ligase enzyme added (B) and recombinant plasmids made (C). (Image by D. Lee)

    The formation of a recombinant plasmid is taking place in a test tube. The cutting and ligating enzymes will work if the right combination of DNA and enzymes is added even if the gene cloner cannot see what is happening inside the tube. Because the resealing relies on chemistry and the chance encounter of sticky ends, a number of different kinds of recombinant plasmids can be made. One common result is the sticky ends from the same plasmid molecule coming together. This will give the same plasmid sequence as the original and is called the nonrecombinant plasmid. Thus the test tube of the gene cloner will contain a combination of recombinant and nonrecombinant plasmids. Their next step is to introduce these plasmids into E.coli bacteria cells.

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    Source : passel2.unl.edu

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