Title: Unit 6 Reading and Multimedia Assignment
Course: SAT 170 Introductory Biotechnology > Section: SAT 170 Introductory Biotechnology

Unit 6 Reading and Multimedia Assignment

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- Unit 6
- Table of Contents (Selected)
- Unit 6 Glossary (Selected)

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“Recombinant DNA Synthesis” by Boundless, CC BY SA 3.0

https://www.boundless.com/biology/biotechnology/dna-and-biotechnology/recombinant-dna-synthesis/
Reading Assignment: Recombinant DNA
Cloning a gene involves cutting DNA using restriction enzymes and uniting it with a cut plasmid, then rejoining the strands with ligase.

To clone a gene, a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes. It is then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a recombinant DNA molecule. Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA.

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single-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. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction. In addition, host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two processes form the restriction modification system. To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone of the DNA double helix. The fragments of DNA generated by this process are called restriction fragments.

Restriction enzymes recognize a specific sequence of nucleotides, called a restriction site, and produce a double-stranded cut in the DNA. While recognition sequences vary between four and eight nucleotides, many of them are palindromic, corresponding to nitrogenous base sequences that read the same backwards and forwards. If many copies of DNA are cut with a restriction enzyme, they will produce identical fragments because restriction enzymes always cut at the same nucleotide sequence.

Restriction enzymes can cut DNA in two different ways. Many make a simple double-stranded cut, giving a sequence what are called blunt or flush ends. Others cut the two DNA strands at different positions, usually just a few nucleotides apart, so that the resulting DNA fragments have short single-stranded overhangs called sticky or cohesive ends. By carefully choosing the appropriate restriction enzymes, a researcher can cut out a target DNA sequence, open up a cloning vector, and join the two DNA fragments to form a recombinant DNA molecule.

Once the sticky ends of recombinant DNA molecules bind to eachother, the new bond must be sealed by repairing the the sugar-phosphate backbone. Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly important in lagging strand DNA replication, as they join the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination and in recombinant DNA technology because the complementary binding between sticky ends will not hold unless the ligase rejoins the two strands.