4.5 DNA Cloning Flashcards
What has our knowledge of DNA biology led to?
an ability to manipulate the genes of organisms
What is a genome?
the complete genetic makeup of an organism
What is genetic engineering?
practice of cloning genes and then using them to alter the genome of viruses and cells
What are the uses of genetic engineering?
ranges from producing a product to treating genetic disorders
What is cloning?
the production of identical copies of an organism through asexual means
Why are human identical twins also considered clones?
because the first two cells of the embryo separated and each became a complete individual
What is gene cloning?
the production of many identical copies of a single gene
Why do biologists clone genes?
- want to produce large quantities of the gene’s protein product such as human insulin
- learn how a cloned gene codes for a particular protein
- use the genes to alter the phenotypes of other organisms in a beneficial way
What is gene therapy?
when cloned genes are used to modify a human
What are transgenic organisms?
organisms with foreign DNA or genes inserted in them
What are transgenic organisms frequently used for?
to produce a product desired by humans
What do most processes rely on to produce cloned DNA?
recombinant DNA technology and the polymerase chain reaction
What is recombinant DNA (rDNA)?
contains DNA from 2 or more different sources such as the human cell and the bacterial cell
What do researchers need to make rDNA?
vector
What is a vector?
a piece of DNA that can be manipulated such that foreign DNA can be added to it
What is plasmid?
a common vector that are small accessory rings of DNA from bacteria that are not part of the bacterial chromosome and are capable of self-replicating
How were plasmids discovered?
by investigators studying the bacterium Escherichia coli
What are the two types of enzymes that are used to introduce a foreign DNA into vector DNA?
restriction enzyme: cleaves the vector DNA
DNA ligase: seals DNA fragments
Why are both DNAs cleaved with the restriction enzyme?
so they can have complementary ends that can anneal
Where do hundreds of restriction enzymes occur naturally?
in bacteria. where they act as a primitive immune system by cutting up any viral DNA that enters the cell
Why are they called restriction enzymes?
because they restrict the growth of viruses
What else can restriction enzymes be used for?
as molecular scissors to cut double-stranded DNA at a specific site
Why is both foreign DNA and vector DNA cleaved with the same restriction enzyme?
a gap exists into which a piece of foreign DNA can be placed if it ends in bases complementary to those exposed by the restriction enzyme
What are “sticky ends”?
the single-stranded but complementary ends of the two DNA molecules
Why are they called “sticky ends”?
they can bind a piece of foreign DNA by complementary base pairing
What do “sticky ends” facilitate?
the insertion of foreign DNA into vector DNA
What is DNA ligase used for?
to seal the foreign piece of DNA into the vector
What do bacterial cells do?
take up recombinant plasmids, especially if the cells are treated to make them more permeable
What happens when the plasmid replicates?
so does the foreign DNA and thus the gene is cloned
What are the steps of gene cloning? (4)
- restriction enzyme cleaves DNA
- DNA ligase seals human gene and plasmid
- host cell takes up recombined plasmid
- gene cloning occurs
Describe cloning a human gene.
- human DNA and bacterial plasmid DNA are cleaved by a specific type of restriction enzyme (ie. human DNA containing the insulin gene is spliced into a plasmid by DNA ligase)
- gene cloning is achieved after a bacterium takes up a plasmid
- if the gene functions normally as expected, the product (ie. insulin) may also be retrieved
What is DNA fingerprinting?
a technique that involves treating the entire genome with restriction enzymes
Why does each person have a unique collection of DNA fragment sizes?
because each person has their own restriction enzyme sites
What is gel electrophoresis?
where an electrical current is used to force DNA through a porous gel material
What is a DNA fingerprint?
each person’s unique collection of DNA fragments are separated according to their size during gel electrophoresis
smaller fragments move farther through the gel than larger fragments and results in a pattern of distinctive bands called a DNA fingerprint
What is short tandem repeat profiling?
the method of choice
STRs are the same short sequence of DNA bases that recur several times as in GATAGATAGATA
Why is STR profiling advantageous?
does not require the use of restriction enzymes and instead, PCR is used to amplify target sequences of DNA which are fluorescently labelled
