Transcript Aim: What are some techniques used in DNA engineering?

Aim: What are some
techniques used in DNA
engineering?
Making cDNA
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Complimentary DNA
(cDNA) is made in vitro.
mRNA from the selected
gene is isolated
Reverse transcriptase
creates a cDNA.
mRNA is degraded
The second strand of
DNA is made.
Nucleic Acid Hybridization
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1) Transfer cells to filter
2) Treat cells on filter to
denature DNA
3) Add a cDNA
radioactive probe;
hybridization will occur
4) expose photographic
film; exposed areas are
colonies with the cloned
DNA
5) compare to original
petri dish.
PCR (polymerase chain
reaction) (in vitro)
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This technique is used
to make many copies
(billions) of DNA
fragments in a few
hours..
It involves heating,
cooling, & replication.
The DNA is incubated in
a test tube with special,
heat-tolerant DNA
polymerase, a supply of
nucleotides, and short
pieces of singlestranded DNA as a
primer.
PCR (polymerase chain
reaction)
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Devised in 1985, PCR has had a major impact
on biological research and technology.
PCR has amplified DNA from a variety of
sources:
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fragments of ancient DNA from a 40,000-year-old
frozen wooly mammoth,
DNA from tiny amount of blood or semen found at
the scenes of violent crimes,
DNA from single embryonic cells for rapid prenatal
diagnosis of genetic disorders,
DNA of viral genes from cells infected with
difficult-to-detect viruses such as HIV.
Gel Electrophoresis
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Gel electrophoresis separates macromolecules
- nucleic acids or proteins - on the basis of
their rate of movement through a gel in an
electrical field
For linear DNA molecules, separation depends
mainly on size (length of fragment) with
longer fragments migrating less along the gel.
After treating long DNA molecules with a
restriction enzyme, the fragments can be
separated by size via gel electrophoresis.
The separated fragments can be recovered
undamaged from gels, providing pure samples
of individual fragments.
Gel Electrophoresis
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In gel
electrophoresis, the
restriction fragments
from two differnt
alleles will produce
different band
patterns, allowing us
to distinguish them
apart.
Southern Blotting technique
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Southern blotting (Southern
hybridization) allows us to transfer the
DNA fragments from the gel to a sheet of
nitrocellulose paper, still separated by size.
Bathing this sheet in a solution containing
a radioactive probe allows the probe to
attach by base-pairing (hybridize) to the
DNA sequence of interest
We can visualize bands containing the label
with autoradiography.
Southern Blotting
Sanger Method
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This method is used to determine the
nucleotide sequence of DNA.
It is similar to PCR, however, special
nucleotides, called dideoxynucleotides, are
used to copy the DNA as tiny fragments.
The order of these fragments via gel
electrophoresis can be interpreted as the
nucleotide sequence.