ch 19 Flashcards
Recombinant DNA technology (genetic
engineering)
– Techniques for locating, isolating, altering, and studying DNA segments
The molecular genetics revolution
Biotechnology: the use of these techniques to develop new
products
Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
- Isolate DNA segment or gene from remaining DNA
- Cut and join DNA fragments—restriction enzymes
- View DNA fragments
- Locate DNA fragments with probes (DNA or RNA
with a base sequence complementary to a
sequence in the gene of interest)
in vivo
– within the cell
Gene cloning with bacteria
in vitro
– outside the cell
PCR is an in vitro technique used for DNA
amplification
Buffers
– helps to create a proper environment/pH for critical components
dNTPs
– the supply of building blocks to build the new DNA strands
MgCl2
is a catalyzer in PCR because it encourages polymerase function
Taq DNA polymerase
– type of DNA polymerase I used for DNA synthesis;
very temperature stable (derived from a thermophilic bacterium)
Primers
– designed to flank the target region and provide a starting point for synthesis. Forward and reverse primers are needed!
Template DNA
- DNA being
replicated.
Some DNA sequences are
– amplified with gene cloning.
– Often used to alter cells so that they have desired
properties or produce substances of commercial value.
*Application: the genetic engineering of plants with
pesticides
Gene cloning
amplifying a specific piece of DNA via a bacteria
cell
Cloning vector
get your gene into cells studying
a replicating DNA
molecule attached with
a foreign DNA fragment
to be introduced into a
cell
Plasmids
circular DNA
molecules that exist
naturally in bacteria
- Transformation of host cells with plasmids
– Chemical treatment
– Physical treatment; i.e. electroporation
What would happen if you forgot to include a selectable marker in your plasmid?
selectable marker: to know which took up resistance
will be impossible to distinguish between transformants (host bacteria with rDNA) and non- transformants.
Microsatellites:
Short tandem repeats (STRs), variable number of copies of repeat sequences
possessed by many organisms
* Detected by PCR
Fragments represented as peaks on a graph
- Homozygotes: single tall peak
– Heterozygotes: two shorter peaks
Sanger’s dideoxy sequencing method
– Based on DNA elongation by DNA
polymerase.
– Uses Dideoxyribonucleoside
triphosphate (ddNTP)
* identical to dNTPs, except lacks a 3′-
OH group, which terminates DNA
synthesis
Microsatellites
Short tandem repeats (STRs),
variable number of copies of repeat sequences
possessed by many organisms
* Detected by PCR
Fragments represented as peaks on a graph
– Homozygotes: single tall peak
– Heterozygotes: two shorter peaks
PCR steps generalized
Goal: To make copies
PCR does this by heating strands to separate DNA without destroying
DNA fingerprinting
many places where
