Gene isolation and manipulation - Overview: isolating and amplifying specific gene fragments
3 important questions on Gene isolation and manipulation - Overview: isolating and amplifying specific gene fragments
Which 2 methods can be applied to obtain the gene of interest?
- In vivo, by tricking the replication machinery of a bacterium into amplifying recombinant DNA containing the gene
- In vitro, in the test tube using the polymerasechain-reaction technique. Both methods employ the basic principles of molecular biology: the ability of specific proteins to bind to DNA and the ability of complementary single-stranded nucleic acid segments to hybridize together (the primer used in the test-tube method).
Which molecular tools (enzymes) do you need when you want to clone a gene?
- Restriction endonucleases: molecular “scissors.” They cut long chromosome-size DNA molecules into hundreds or thousands of fragments of more manageable size.
- Ligase
- DNA polymerase
- Primer
Why do you need to convert the mRNA into CDNA?
Complementary DNA (cDNA) is a DNA version of an mRNA molecule. Researchers use cDNA rather than mRNA itself because RNAs are inherently less stable than DNA. Moreover, RNA cannot be manipulated by the enzymes available for DNA cloning, and techniques for routinely amplifying and purifying individual RNA molecules do not exist.
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