P V 9 2 A N D P C R Flashcards
it allows a researcher to make a large number of copies
of a particular stretch of DNA.
PCR
Medicine: PCR can be used to detect specific _______. One example of this would be finding virus DNA that has been inserted into a person’s cells. HIV, the virus that causes ___, is one example of a virus that can insert its DNA into a cell’s chromosomal DNA.
DNA sequences
AIDS
_____ allows you to specifically find and copy the virus DNA sequence amidst all that excess information. If you do PCR and find HIV DNA
in a cell, you’ve proven that the cell has been infected with the virus. In addition, you can use the virus DNA to learn more about the virus.
PCR
\_\_\_\_\_is widely used as an all-around molecular biology technique. Anybody who works with DNA eventually wants to copy some DNA, and \_\_\_has become the easiest, fastest, and cheapest way to do it.
PCR
The DNA region, or ____, that you’ll copy is called ____. The PV92 locus is part of an intron on _____
locus
PV92
chromosome 16
The thing that makes PV92 useful for _______ is that it’s variable. Everybody has the PV92 locus, but some people have an extra stretch
of repetitive DNA (called an ______) in it
DNA fingerprinting
Alu repeat
The _______ is a stretch of 300 nucleotides that occurs over and over at various locations in the human genome. Apparently, this
segment of noncoding DNA has been duplicated many times during the evolution of primates, and inserted into the genome at
many locations.
ALU repeat
There are only two common PV92 versions, or alleles, in the human population – the short one, with ______, and the long one,
with the _______.
no Alu repeat
Alu repeat
Since everybody has two copies of chromosome 16 (one from mom and
one from dad), everybody has two copies of the ______. Therefore, you could possibly get both a short and a long PV92 PCR product
from one person.
PV92 locus
If you have two copies of
the same allele (+/+ or -/-),
you’re _______`.
homozygous
If you have two different alleles (+/-), you’re _______
heterozygous.
In the PCR for this lab, the product of the + allele will be \_\_\_\_\_ base pairs long, while the product of the – allele will be \_\_\_\_\_ bp.
941
641
If your genotype is \+/+, you’ll see one \_\_\_\_\_\_ band on the gel. If your genotype is -/-, you’ll see only a \_\_\_\_\_ band on the gel. If your genotype is +/-, you’ll see a \_\_\_\_\_\_
941 bp
641 bp
short band and a long one.
In real DNA fingerprinting, you’d have to look at many ____
together before you could definitely say that two DNA samples are the same or are closely related.
loci
The tools used for copying DNA in PCR are the same tools used by cells in copying their own
DNA: a ______, some _______, _____, and the DNA to be copied (called the template). The name Polymerase Chain Reaction comes from the fact that a DNA polymerase is used to copy fragments of DNA
over and over in a chain reaction.
DNA polymerase
nucleotides
primers
_______, or strand separation: the two strands of the DNA double helix are separated. In living cells, the strands are separated by an enzyme (______). In PCR, the two strands
are separated by simply heating the DNA to 90 or so. This breaks the hydrogen bonds joining the two strands, but leaves the
covalent bonds of each strand intact.
Denaturing
helicase
_______: in this step, the primers bind to the DNA. In living cells, the primers are made of RNA are synthesized right on the
DNA by an enzyme (_____). In PCR, the primers are artificial and are designed by the researcher to base pair with a specific region
of DNA.
Primer annealing
primase
_______: in this step, the polymerase goes to work. The polymerase used in PCR, like any
DNA polymerase, can only work by adding new nucleotides to the 3’ end of an existing DNA strand. The polymerase copies DNA starting at the primer. Extension happens fastest at the enzyme’s optimal temperature.
Extension
The DNA polymerase most commonly used in PCR is called ___, which is short for ______, the heat-loving bacterium from
which the enzyme was first isolated.
Taq
Thermus aquaticus,
______: to make PCR happen, you repeat the above three steps over and over. Repeating
the denaturing, annealing, and extension steps is done by controlling the temperature.
The temperature can be controlled by a thermal cycler, or PCR machine, which can be
programmed to put a set of PCR tubes through a programmed set of temperature cycles. (A cycle consists of denaturing, annealing, and extension. A complete reaction might require _____.)
Repeat
25 cycles
_____: PCR can amplify DNA starting from a tiny quantity of template – as little as a single molecule. This is true because PCR is a chain reaction, making DNA copies and then
making copies of the copies.
Sensitivity
______: PCR can be used to copy exactly the DNA segment you want, even if you start
with a complex mixture of template DNA. This is true because each primer is complementary to only one stretch of nucleotides on the
template. PCR conditions can be controlled so that the primer will bind only where it is
exactly complementary to the template.
Specificity
DNA doesn’t need to be very pure for PCR, but you need to get rid of two things:
_________, and ________ that would interfere with the PCR
enzyme. You can achieve these two goals by simply boiling your cells with a product called ___.
enzymes that might damage DNA
cations like Mg++
Chelex
Boiling kills the enzymes and lyses
the cells. ___ is a solid resin that is negatively charged, so it binds cations. It isn’t water soluble, so it will be a powder in the bottom of your micro tube, pulling away
harmful ions and leaving you with ready-touse DNA.
Chelex
