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Genetic Engineering
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Genetic engineering is also referred to as
recombinant DNA technology – new
combinations of genetic material are produced
by artificially copying a piece from one organism
and joining this copy with DNA of another
organism (this works because the genetic code
is universal) – the following must be
accomplished to create new genetic
combinations:
1.
2.
3.
4.
locate a specific gene in the donor cell
isolate this gene in a piece of donor DNA
modify the donor DNA in a highly selective way
transfer the modified donor DNA into host cells in a
way that the gene will be expressed
Locating and isolating genes – can be
accomplished using various techniques
• Genetic probes – can be used if at least part
of the DNA base sequence in the required
gene is known
– probe consists of a single strand of DNA that
contains the known sequence of bases
– probe is labeled with a radioactive marker
– bases in genetic probe combine with the
complementary bases on the donor DNA,
revealing position of gene
• Reverse transcription – can be used when cells
produce large amounts of the particular polypeptide
1. cells will contain mRNA for polypeptide
2. mRNA is isolated and can be used to make the
complementary DNA through reverse transcription –
reverse of normal transcription
3. requires DNA nucleotides and enzymes called reverse
transcriptases
4. after mRNA has been used to make DNA, mRNA is
removed and the complementary strand of DNA is made
by adding the enzyme, DNA polymerase and more DNA
nucleotides
5. the result is a double-stranded DNA molecule identical to
the original DNA molecule
6. reverse transcriptases were first obtained from
retroviruses
• Artificial DNA synthesis – a piece of DNA
coding for a particular protein can be
made artificially if the base sequence is
known
– this can be obtained from knowing the amino
acid sequence of the protein
– useful for small proteins
Enzymes – two important enzymes used in genetic
engineering are restriction endonucleases and DNA
ligases
• Restriction endonucleases – cut DNA at specific
points
– cut ends are called “sticky ends” because they have
exposed bases that will form hydrogen bonds with
complimentary sticky ends from other DNA molecules cut
by same restriction endonuclease
• DNA ligases – these enzymes stick lengths of DNA
together
• genetic engineers are able to use both enzymes to
“cut and splice” DNA
A DNA vector is used to transfer DNA from one cell to
another
• most commonly used vector is a plasmid – small ring of
DNA contain in some bacteria
• separate from main chromosome
• replicate independently
• can be passed from one bacterial cell to another (occurs
naturally)
• transfer to another cell can be encouraged artificially
• restriction enzymes and DNA ligase are used to cut open
a plasmid so that donor DNA (gene) can be inserted
• modified plasmid is transferred to another living cell
(usually a bacterial cell)
• this process is possible because of the universality of the
genetic code
Genomic Libraries – (total DNA of a cell is referred
to as a genome)
• genomic library – a collection of DNA fragments
that are more or less representative of all the
DNA in the genome
• each fragment is spliced into a plasmid, which is
usually contained inside a bacterial cell
• restriction enzymes may divide a single gene
into more than one fragment, therefore some
genes in a genome library may be divided up
among two or more clones (bacterial cell
containing plasmid)