midterm study guide Flashcards
(44 cards)
what is the purpose of using a micropipette
micropipettes are standard labaratory equipment used to accurately measure and transfer small volumes of liquids .
what 2 micropipettes use the same tips
the middle sized tips are for both p20 and p200s.
what are homologous genes
homologous genes are two or more genes that descend from a common ancestral DNA sequence.
orthologous genes vs paralogous genes
orthologous genes result when sequences diverge from the ancestral gene after speciation
paralogous genes result when gene duplication followed by sequence mutation happens within a species.
why is GADPH genetic code for a particular protein of interest
GADPH is a crucial enzyme in glycolysis and thus the DNA coding for the enzyme is considered a housekeeping gene
GADPH is abundant in cells and can be purified
what are the 2 major domains of GADPH
NAD+ binding domain
C terminal doman with dehydrogenase activity
explain what happens in the first round of PCR
in the first round a mixture of primer sequences called degenerate primers is used to increase the odds, that at least one version is able to stimulate the copying of the region of interest.
explain the second round of PCR
the second round of PCR uses nodegenerate primers known as the nested approach to yield many copies of a single or a few uniform sequences .
what is the purpose of agarose gel electrophoresis
To be able to identify the PCR products of different lengths by their variability in gene structure.
why is nucleic acid extraction essential
nucleic acid extraction allows the isolation of genomic DNA from experimental tissue.
explain the overview of isolation of gDNA from plants
- harvest cells: harvest plant tissue
- physically disrupt plant cells from plant tissues
- lyse cells to disrupt cell membrane
4.remove insoluble cell components, such as the remains of cell wall and membranes by centrifugation.
- Purify DNA by removing contaminating proteins and RNA .
what does PCR stand for and what’s the purpose of PCR ?
Polymerase chain reaction is a technique for rapidly creating mutiple copies of a segment of DNA utilizing repeated cycles of DNA synthesis.
Since the goal of PCR is to amplify the DNA region of interest,
describe the DNA primers in PCR.
the DNA primers should be exactly complementary to the target sequence they should bind to; this type of primer design is possible when the exact sequence in the template is already known.
what are the componenets needed for PCR
Taq DNA polymerase (or another thermally stable DNA polymerase)
* Primers — synthesized to complement a specific region on the template DNA. The two
primers in a pair are designed to anneal to opposite ends of the region of interest.
* Nucleotides — the four individual bases in the form of deoxynucleoside triphosphates
(dNTPs), which allows them to be added to a DNA polymer. The dNTP mixture includes
the same amounts of dATP, dTTP, dGTP, and dCTP
* Reaction buffer
what are the steps of PCR
The first step of the PCR reaction is the denaturation step. the first step of PCR is uncoiling and separating the two strands
of the template DNA.
to allow the primers to anneal to
the separated template strands. The temperature at which the primers anneal to the template DNAdepends on several factors, including primer length, the G–C content of the primer, and the
specificity of the primer for the template DNA.
step, but they can also result in amplification
of DNA other than the target. In the annealing step, the two original strands may re-anneal to eachother, but the primers are in such excess that they outcompete the original DNA strands for the
binding sites.
The final step is extension, in which the reaction is heated to 72°C, the optimal temperature for
Taq DNA polymerase to extend the primers and make complete copies of each template DNA
strand. At the end of the first PCR cycle (one round of denaturation, annealing, and extension
steps define one cycle), there are two new strands for each original double-stranded template,
describe the cycles of PCR
During the first cycle, primers anneal to the
original template DNA strands at opposite ends and on opposite strands. After the first cycle, two
new strands are generated that are shorter than the original template strands but still longer than the target DNA. It isn’t until the third PCR cycle that fragments of the precise target length are
generated
describe primers
PCR is the design of the primers, which will determine
whether or not the correct piece of DNA is amplified. Primers are short, single stranded
oligonucleotides, designed to bind to the DNA template strands
at the ends of the sequence of interest. two different primers are needed, one for each
of the complementary strands of template DNA, flanking the ends of the region of double stranded
DNA to be copied. Primers are defined sequences of nucleotides, designed based on the known
sequence of the target DNA.
WHAT IS THE ROLE OF A PRIMER
primers will determine whether or not the correct region of template DNA is amplified.
Once primers are designed, then it is possible to complete a PCR experiment to evaluate gDNA
isolated from organisms of interest.
FACTORS CONSIDERED IN A PRIMER DESIGN
LENGTH
MELTING TEMPERATURE OF PRIMERS
ANNEALING TEMPERATURE
GC CONTENT
GC CLAMP
INTRA COMPLEMENTARITY PRIMERS
ADVANTAGES AND DISADVANTAGES OF DEGENRATE PRIMERS,
Degenerate primers are beneficial because they increase binding to the target region during PCR,
but they have drawbacks. First, they decrease the concentration of specific oligos available for
amplification, which reduces the efficiency of the PCR. Second, they increase the chance of
amplification of nonspecific/unwanted PCR products.
WHAT DISTINGUISHES THE SECOND ROUND OF PCR ?
The second round of nested PCR does not use degenerate primers, which increases its specificity
for the single gene of interest.
what happens in nested pcr
Nested PCR uses
two sequential sets of primers. The first primer set binds to sequences at the edge of the target
DNA, as expected in standard PCR; however at least one (or several) of the options may also bind to other areasof the template gDNA.
The second primer set binds to sequences in the target DNA that are within
the portion amplified by the first set (that is, the primers are nested because they will anneal just
inside the first set). Thus, the second set of primers will direct amplification of target DNA that is
just slightly shorter in length than the products of the first reaction.
advantages of PCR
One advantage of nested PCR
is that if the first primers bind to and amplify any unwanted DNA sequence, it is very unlikely that
the second set of primers will also bind within the unwanted region.
A second advantage of nested PCR is that the initial PCR step enriches the pool of potential targets
for the second set of nested primers.
WHY DOES GDNA NEED TO BE DILUTED.
Following exonuclease I treatment and inactivation of the enzyme, the PCR products from the
gDNA templates generated in the first round of PCR need to be diluted. The PCR products from
the initial round contain a high proportion of GAPC-like sequences relative to the total amount of
gDNA. By diluting the gDNA, it is even less likely that the gDNA will be a template for
contaminating PCR products when using the nested primers in the next round of PCR.
