Download PCR reading answers

LHWHS AP Biology
NAME_______________________
PCR / DNA tech reading
p. 174
1. What is pharming ?
genetically engineering farm animals to produce pharmaceuticals
p. 175
2. What are oligionucleotides ?
short, single stranded pieces of DNA
3. In what year did Kary Mullins win the Noble Prize for 'inventing' PCR ?
1993
4. What are primers ?
oligionucleotides (short, single stranded pieces of nucleic acid) designed to
match and bind with a targeted segment of the genome
5. What does it mean when we say that primers "provide the specificity of
PCR" ?
primers bind with the targeted or selected region of DNA that is to be
amplified using PCR
p.176
6. What is unique about the DNA polymerase enzymes used in PCR ?
The polymerase enzymes used in PCR are thermophillic (heat resistant).
This property allows the DNA to be denatured (separated) using heat while
being replicated using thermophillic polymerase enzymes.
7. How is DNA denatured prior to replication using PCR ?
heat to around 94 degreed Celsius
Is the sequence of nitrogen bases changed as a result of denaturing ?
NO, NO, NO, NO
8. What type of bonds allow primers to anneal to the target sequence ?
hydrogen bonds hold strands of the double helix together ; logically...it
follows that hydrogen bonds are formed when primers anneal to the target
sequence.
How are environmental conditions changed when allowing primers
to anneal with the target sequence ? cooled ;
change from 94 degrees C to 50 or 60 degrees C
9. Name the enzyme responsible for 'extension' of the primers.
(thermophillic) DNA polymerase
p.177
10. What is an amplicon ?
the product of PCR ; the replicated sequence of targeted nucleotides which
has been copied a million (or sometimes a billion) times
11. Where does in vivo replication occur ?
inside a living cell (in vivo...in life vs in vitro.....in glass)
12. Briefly explain the role of each enzyme for in vivo replication.......
topoisomerase - stabilizes the DNA helix ahead of the replication
fork ; it does allow for some unwinding of the double helix in a
controlled manner during replication or transcription....
.....literal translation = "enzyme that keeps the top the same"
helicase - breaks hydrogen bonds in the replication fork , "unzips" the
DNA double helix
primase - enzyme that makes primers by joining single stranded
nucleotides together
DNA polymerase - enzymes which 'extend' the primer for both leading
and lagging strands which contain discontinuous fragments
(Okazaki fragments) ; provide environment in which nucleotides
can bind to complementary strand ; nucleotides are made
as a result of cell metalbolism (ATP, GTP, CTP, TTP)
ligase - enzyme which creates an environment that allows for the
joining of Okazaki fragments (discontinuous fragments
from the lagging strand)
p.178
13. What is the difference between gDNA and cDNA ?
gDNA is genomic DNA. Genome is often used to refer to all of an organism's
genes or sequence of nucleotides (nitrogen bases).cDNA is complementary
DNA. It is also fair to think of cDNA as copied DNA. Often the product of
using reverse transcriptase is cDNA. The enzymes from retro-viruses create
an environment in which mRNA is used to make a complementary (or copied)
DNA strand.
14. What is the role of dNTP's (deoxyribonucleoside triphosphates) in a PCR
reaction ? provide both an energy source and a nitrogen base for building the
new strands of DNA
15. What salt (ionic compound) is an essential co-factor for DNA polymerase?
(hint.....co-enzymes (organic molecules); co-factors (usually contain a metal))
magnesium chloride (MgCl2)
p.179
16. What is a thermo-cycler ?
(thermal cycler) machine that cycles through various temperatures (heating
and cooling) in an effort to denature DNA, to allow primers to anneal, and to
allow a mixture of polymerase and dNTP's to build copied strands of DNA
17. In the movie "The Human Spark", neanderthal DNA was amplified using
PCR. The lab technician mentioned that it is not uncommon for people to
mistakenly amplify their own DNA along with DNA from neanderthal bone
fragments. Why is PCR very "prone to contamination" ?
It could be easy for a pieces of cellular debris and/or DNA to be accidentally
transferred to the microtubes. Using gloves, UV light, filtered pipet tips, and
screw-cap tubes will minimize the level of contamination.
18. What is included in the "master mix" ?
thermophilic DNA polymerase, dNTP, primers, buffer
What is the master mix "added directly to" ?
template DNA
p.180
19. If nucleotides produced from PCR are often smaller than plasmid DNA,
then the agarose gel used to analyze the products for PCR should
be_more__concentrated than gels used to analyze plasmid DNA.
(more or less)
more concentrated agarose---> smaller pore size
p.181
20. What is reverse transcriptase ? Where does it come from ? What is it
capable of making ?
Reverse transcriptase is an enzyme that transcribes mRNA into DNA. This
enzyme comes from retro viruses. The resultant DNA is called cDNA
(complementary or copied) that is often used as the template DNA for PCR.
p.183
21. What determines the choice of primer to be used in PCR ?
The nitrogen base sequence at the ends of the target region
p.185
22. What can DNA microarrays be used to determine ?
Microarrays can be used to determine which genes in a cell are turned on and
off
List two examples indicating why this info is useful ?
In general, this info helps us compare abnormal and normal tissue
(i.e. cancer vs. healthy). This info can also be helpful in determining how
cells will respond to different drugs.
23. In step one, what is each dot on a printed microarray ?
Each dot is a gene which contains multiple copies of single strands of DNA.
Often, these multiple copies of a gene are produced using PCR.
24. In step two, what does it mean for the microarray to be incubated ?
Incubated simply means mixing together under controlled conditions. cDNA
from abnormal tissue and cDNA from healthy tissue is mixed with the single
strands of DNA (genes) on the microarray.
25. In step three, how can someone tell if the gene is expressed ?
Generally, cDNA from healthy tissue is tagged with green markers (stains).
cDNA from abnormal tissue is tagged with red markers (stains). As the
cDNA binds with the single strands on the microarray, the dots become red,
green, or yellow based on which cDNA is able to bind with the 'dots'. If no
binding occurs, then the 'dot' does not change color. Level of expression is
associated with the color change of the 'dot'. In other words, if cDNA from
the tissue sample binds with the genes on the dot, then there is a higher
likelyhood that the gene is expressed. It is important to note that the cDNA
is made from mRNA isolated in each tissue sample. Reverse transcriptase is
used to transcribe mRNA into cDNA. Since the cDNA is made from previously
transcribed mRNA, it is fair to think of the 'sticking' to the dot as a measure
of gene expression.
p.186
26. What process is used to amplify the single standed DNA used for each
spot of the array ? PCR
27. Briefly explain how healthy and cancerous cell gene expression is
compared using a microarray.
(See #25) Once again, the color of the dot is an indicates if the gene was
used to make mRNA. Bioinformatics is the study of gene expression and the
pathways used by the cell to express (make protein from DNA) the gene.
In my humble opinion, using this information is the future of biology and
medicine.
p.187
28. Briefly, mention the value of PCR when studying genetically modified
foods......both in the field where food is grown and in the store where GM
food is sold.
Corn and soybeans have been genetically modified with soil bacterium plasmids
The gene products of these plasmids are toxic to insect larvae. In other
words, the genetically modified crops can become resistant to pests. Other
genetically modified crops are resistant to herbicides and frost.
PCR can help identify if genetically modified crops are spreading outside of
their defined boundaries in the field. PCR is also used in food testing to
ensure that food meets governmental regulations.