BCMB 406B Midterm 1 lab 1
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1. PCR allows for: amplification of minute amounts of template
DNA
can generate thousands or millions of copies of
that sequence
2. What is one of the most well designed primers
important parameters for a
successful PCR?
3. Name 3 variables that influ- 1. quality of template DNA
ence the success of a PCR 2. cycling conditions
reaction 3. specificity and stability of PCR primers
4. The target sequence de- 1. primer length
termines what criteria in 2. % GC content
primer design for PCR? 3. melting temperature
=modifications to one of these will change the
others, meaning they are linked
4. It will also influence the likelihood of primers
to form hairpins and dimers and cross-dimers
5. What is the standard A) initial denaturation for 1-5 minutes at 94 degC
A) denaturation tempera- (influenced by nature of template DNA)
ture B) Typically 30 seconds at a lower temperature
B) Annealing temperature (influenced by desired specificity)
C) Elongation temperature C) 30 seconds per 1000 bp at 74degC
(influenced by product length)
It is also common to have a final extension of
5-15 minutes
6. The nature of template Plasmid DNA: takes about 1 minute to denature
DNA influences denatura- Chromosomal DNA: takes 3-5 minutes
tion times during PCR cy-
cles: =duration of denaturing steps typically 30-45
How does the length of de- seconds
, BCMB 406B Midterm 1 lab 1
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naturation relate to the na-
ture of template DNA?
7. The number of cycles in a 1. The amount of template DNA available at the
PCR experiment depends beginning of a PCR
on what two things? 2. What will be done with the DNA product after
reaction completion
Fewer cycles = less final product
Fewer cycles = may reduce background prod-
ucts
8. PCR reactions where the 1. Between 1x10^5 to 1x10^6 number of tem-
template DNA is added in plate DNA molecules
a puridied form, typically 2. Between 20-30 cycles
starts with between _____ -
___ molecules of template (page 1-3)
DNA
Therefore in a PCR reac-
tion you would expect from
___ - ___ cycles.
9. site-directed mutagenesis The ability to make precise genetic mutations
in any location of a DNA molecule, in a precise
manor
=allows for the ability to make substitutions,
insertions and deletions in a target DNA se-
quence
10. ______ methodology al- Site directed mutagenesis
lows for the study of com-
plex
*cellular regulation of
genes
*the role of specific amino
acid residues in protein
structure, catalytic func-
tion and ligand binding ca-
, BCMB 406B Midterm 1 lab 1
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pacity
*mechanisms involved in
infectious and genetic dis-
ease
11. The essential components 1. DNA template
of DNA synthesis reac- 2. primer(s)
tions are: 3. dNTPs
4. DNA polymerase
12. Several PCR-based meth- 1. over-lap extension PCR
ods for performing SDM 2. megaprimer PCR
have been developed, what 3. inverse PCR
are they?
13. Advantages: Over lap extension PCR
1. good mutational effi-
ciency
2. large deletions possible
3. Long primers are not
necessary
4. Useful for point muta-
tions, deletions and gene
infusions
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