Theme 4: DNA Replication and Mitosis - Module 3: Applications of DNA Replication Flashcards
what technique did Kary Mullis develop?
the ability to amplify DNA in a rube vie polymerase chain reaction (PCR) technique
what did this technique allow scientists to do?
to be able to copy (amplify) millions of copies of DNA from very small starting samples
DNA in a tube has revolutionized the world of cellular and molecular biology in what ways?
- has shed light on diagnosis of genetic defeats
- detection of viral DNA in cells
- producing large amounts of DNA from fossils containing trace amounts of DNA
- being able to link specific individuals to DNA samples during forensic investigations
how is a PCR set up?
a sample of DNA is placed into a tube containing a buffered solution with essential ions and salts, along with a pair of short single-stranded DNA primers (usually 15-30 nucleotides in length)
what do the primers do?
bind in a complementary manner to specific regions of the template DNA and serve as starting points for DNA copying
since this is a cell-free system what else is also added within the tube?
free deoxyribonucleotides (dNTPs)
when are the dNTPs utilized?
during the replication process
what does PCR not require?
the multiple enzymes that cells utilize to unwind and stabilize the DNA double helix
where are the tubes placed instead?
in a thermocycler machine
what happens with the thermocycler machine?
it goes through various phases of heating and cooling in automatic programmed steps to facilitate the DNA replication process over various repeated cycles
how many key DNA replication enzymes are still required?
one
what is it?
a special DNA polymerase
what is special about this DNA polymerase?
it is tolerant to high temperatures
why is it added to the tube?
to catalyze the polymerization of each daughter strand within the tube with each replication cycle
what is an example of this type of heat-tolerant DNA polymerase?
Taq polymerase
where was this first isolated from?
the bacterial species Thermus aquaticus - adapted to live in hot springs with temperatures as high as 95 degrees celsius
how many key stages are involved in a PCR reaction
three
what are they
denaturation, annealing, and extension
what does this three step cycle bring?
a chain reaction that produces an exponentially growing population of identical DNA molecules
the types of DNA molecules were referring to depends on what?
the types of primers that are designed
most researchers have a sequence of a gene or DNA segment that they wish to replicate or cone, thus how will researchers design primers?
design primers to bind to or anneal to their complementary sequence on either side of the DNA sequence of interest on the DNA template strands
how must the DNA double helix be at the start of a PCR?
unwound
how is this facilitated?
by a high temperature stage of the reaction
what happens during the denaturation stage?
the reaction mixture is heated to separate the strands of the double-stranded DNA
what will then happen with the thermocycler?
it will cool the solution
what does this allow?
allows the two primers to anneal to their complementary sequences on the DNA template strands on either side of the DNA sequence of interest during the annealing phase
where will the primers bind?
on opposite strands at each end of the target sequence
when does the heat-stable DNA polymerase extend and polymerize the daughter strands?
during the extension phase
what is used?
four dNTPs, starting from the primers and extending the daughter strand in a 5’ to 3’ direction
what does each complete cycle result in?
two double stranded helices containing the desired target sequence portion of the original template DNA
summarize the denaturation step:
a solution containing double-stranded DNA (the template duplex) is heated to separate the DNA into two individual strands
summarize the annealing step:
when the solution is cooled, the two primers anneal to their complementary sequence on the DNA template strands
summarize Extension:
DNA polymerase synthesizes new DNA strands (complementary to the template) by extending primers in a 5’ to 3’ direction
are PCR reactions fast and specific for the DNA sequence that is replicated
yes
with each successive cycle the number of replicated DNA molecules with the same sequence as the parent template duplex is what?
double
what is present after the first cycle of PCR?
two copies of the template duplex - each consisting of one new and one old DNA strand
with each cycle what do the newly synthesized DNA segments serve as?
templates in later cycles
after the second PCR cycle how many copies would be present?
4
what equation can be used to represent the number of copies of the template DNA target sequence
2^n (n=cycles)
what is essential for many of the applications of PCR?
the huge amplification
do the tubes look different after removing them from the thermocycler?
no
what will be in the tube if the PCR was successful?
millions of molecules of amplified segments of DNA
what do researchers use in order to visualize DNA molecules?
gel electrophoresis
what does gel electrophoresis do?
general technique used to separate DNA fragments from other sources, not just from PCR
what else can gel electrophoresis be used to separate?
other macromolecules including RNA and proteins, all on the basis of their rate of movement through an agarose gel in an electric field
where are molecules loaded during DNA gel electrophoresis?
into wells of a porous gel
where do the molecules travel? how?
- along the length of the gel
- because of an electrical field that is applied along the length of the gel
what are the different charges established and where are they located?
positive at one end of the gel and negative at the other end of the gel
what is the charge on DNA and RNA? Why?
- negatively charged
- due to ionized phosphate groups along the phosphodiester backbone
where will they be attracted towards?
attracted towards the positively charged (anode) end of the gel
what also affects how far through the gel the molecules will travel?
size of the molecule
the molecules that travel through the gel at the highest speed tend to be what size?
the smallest molecules
what is the travelling speed and distance of larger molecules?
- slower speed
- smaller distanced along the gel compared to smaller molecules
what is the range of nucleotides that gel electrophoresis can separate?
several hundreds of nucleotides to over 10, 000 nucleotides
what are the PCR results typically?
the amplification of just a single size of DNA molecule
what is it also possible to utilize gel electrophoresis for?
to separate and visualize a DNA sample containing a mixture of DNA fragments of different sizes
what is also loaded onto the gel electrophoresis along with the DNA samples?
a standardized ladder