8 Flashcards
(47 cards)
biotech
—the use of microorganisms to make practical
products
mutagens
Mutagens are physical and chemical agents that produce
mutations (changes in nucleotide sequence of a genome). Scientists
deliberately use mutagens to create changes in microbes’
genomes so that the microbes’ phenotypes are changed. They
then select for and culture cells with characteristics considered
beneficial for a given biotechnological application.
cDNA
Reverse transcriptase creates a flow of genetic information in the opposite direction from the
flow in conventional transcription: it uses an RNA template to
transcribe a molecule of DNA, which is called complementary
DNA (cDNA) because it is complementary to an RNA template.
Because hundreds to millions of copies of mRNA exist for every
active gene,
it is frequently easier to produce a desired gene by
first isolating the mRNA molecules that code for a particular
polypeptide and then use reverse transcription to synthesize a
cDNA gene from the mRNA template.
probes
Probes are nucleic acid molecules
with a specific nucleotide sequence that have been
labeled with radioactive or fluorescent chemicals so that
their locations can be detected. The use of probes to locate
specific sequences of nucleotides is based on the fact that
any given nucleotide sequence will preferentially bond to
its complementary sequence.
antiesnse
Antisense nucleic acid molecules have nucleotide
sequences that bind to and interfere with genes and
mRNA molecules.
naming restriction enzyems
Researchers name restriction enzymes with three letters (denoting
the genus and specific epithet of the source bacterium)
and Roman numerals (to indicate the order in which enzymes
from the same bacterium were discovered). In some cases, a
fourth letter denotes the strain of the bacterium.
restrictione nzyems
Such
enzymes cut DNA molecules and are restricted in their action—
they cut DNA only at locations called restriction sites. Restriction
sites are specific nucleotide sequences, which are usually
palindromes3
—they have the same sequence when read forward
or backward.
In nature, bacterial cells use restriction enzymes
to
protect themselves from phages by cutting phage DNA into
nonfunctional pieces. The bacterial cells protect their own DNA
by methylation4
of some of their nucleotides, hiding the DNA
from the restriction enzymes
sticky ends
makes staggered cuts of the two strands of DNA, producing
fragments that terminate in mortise-like sticky ends.
Each sticky end is composed of up to four nucleotides that
form hydrogen bonds with its complementary sticky end
(Figure 8.2a). Scientists can use these bits of single-stranded
DNA to combine pieces of DNA from different organisms
into a single recombinant DNA molecule
blunt ends
when rest enzymes cut
both strands of DNA at the same point. It is more difficult to make recombinant
DNA from blunt-ended fragments because they are not
sticky, but they have a potential advantage—blunt ends are
nonspecific. This enables any two blunt-ended fragments,
even those produced by different restriction enzymes, to be
combined easily
vectors
To deliver a gene
into a cell, researchers use vectors, which are nucleic acid molecules,
such as viral genomes, transposons, and plasmids
Genetic vectors share several useful properties:
Small enough to manipulate in a lab
Survive inside cells
Contain recognizable genetic marker
Ensure genetic expression of gene
Generally, __ and __ are able to carry __
genes than can __.
Generally, viruses and transposons are able to carry larger
genes than can plasmids.
process to produce vector
After a given restriction
enzyme cuts both the DNA molecule containing the gene
of interest (in this example, the human growth hormone gene)
and the vector DNA (here a plasmid containing a gene for antibiotic
resistance as a marker) into fragments with sticky ends 1 ,
ligase anneals the fragments to produce a recombinant plasmid
2 . After the recombinant plasmid has been inserted into a bacterial
cell 3 , the bacteria are grown on a medium containing
the antibotic 4 ; only those cells that contain the recombinant
plasmid (and thus the human growth hormone gene as well) can
grow on the medium
gene library
a collection
of bacterial or phage clones—identical descendants—each
of which contains a portion of the genetic material of interest. In
effect, each clone is like one book in a library in that it contains
one fragment (typically a single gene) of an organism’s entire
genome.
generate gene library
isolate genome of organism. using restriction enzymes
to generate fragments of the DNA of interest and then using
ligase to synthesize recombinant vectors. They insert the vectors
into bacterial cells, which are then grown on culture media.
pcr
The polymerase chain reaction (PCR) is a technique by which
scientists produce a large number of identical molecules of DNA
in vitro. Using PCR, researchers start with a single molecule of
DNA and generate billions of exact replicas within hours.
pcr steps
denaturation, primig, extneison
denaturation
Exposure to heat (about 94°C) separates the
two strands of the target DNA by breaking the hydrogen
bonds between base pairs but otherwise leaves the two
strands unaltered.
priming
A mixture containing an excess of DNA primers
(synthesized such that they are complementary to nucleotide
sequences near the ends of the target DNA), DNA
polymerase, and an abundance of the four deoxyribonucleotide
triphosphates (A, T, G, and C) is added to the target
DNA. This mixture is then cooled to about 65°C, enabling
double-stranded DNA to re-form. Because there is an excess
of primers, single strands are more likely to bind to a primer
than to one another
extension
Raising the temperature to about 72°C increases
the rate at which DNA polymerase replicates each strand to
produce more DNA
thermocycler
The process can be automated using a thermocycler, a device
that automatically performs PCR by continuously cycling all
the necessary reagents—DNA, DNA polymerase, primers, and
triphosphate deoxynucleotides—through the three temperature
regimes.
electrophoresis
a technique that involves
separating molecules based on their electrical charge, size,
and shape.
