midterm 15% Flashcards
- A molecular biology technique which simply makes many
copies of specific DNA or genes which takes place in vitro,
laboratory, or test tube, rather than in the cell.
POLYMERASE CHAIN REACTION
- PCR relies on thermostable DNA Polymerase, called
Taq Polymerase
- Heat the reaction strongly to separate, or denature,
the DNA strands.
DENATURING
This provides single-stranded template for the next step,
annealing.
denaturing
- DNA synthesis requires that the template DNA be ______
stranded.
single
- The hydrogen bonds between the complementary bases in the double strands of the template DNA are broken by
heating at what temp??
94° to 95°C for 30 to 60 seconds.
Cool the reaction so the primers can bind to their
complementary sequences on the single-stranded
template DNA.
annealing
oTemperature is ?? to allow primers to base
pair to complementary DNA template.
increased or decreased
decreased
- After denaturation, the temperature is lowered typically
between __°C to __°C for 30 to 60 seconds
47°C to 60°C for 30 to 60 seconds
The ________ temperature of a DNA molecule is described as the temperature at which half of the DNA strands are in a
single-stranded (ssDNA) state
melting
- Raise the reaction temperatures so Taq polymerase
extends the primers, synthesizing new strands of DNA.
extension
The temperature is raised, typically to __°C, to allow the
DNA polymerase to make a complimentary copy of template
starting from the primers.
72 deg
- A typical PCR incudes _____ cycles of amplification, with a
final extension cycle of 10 minutes at 72°C, followed by
incubation at 4°C.
25-40
- A typical PCR incudes 25-40 cycles of amplification, with a
final extension cycle of __ minutes at 72°C, followed by
incubation at 4°C.
10mins
Initial denaturation TEMP/TIME:
95°C, 5 min
Denaturation TEMP/TIME:
94°C, 1 min (25-40 cycles)
- It is a technique in a conventional PCR procedure to
visualize or check if the genes are amplified.
GEL ELECTROPHORESIS
- Gel electrophoresis is a technique in which fragments of
DNA are pulled through a gel matrix by an electric current,
and it separates DNA fragments according to ?
SIZE
DNA of interest called
template DNA
It is extracted
from the sample and it contains the target sequence.
TEMPLATE DNA
Before starting PCR, the purity of the DNA of interest
should be checked using ?
spectrophotometer.
The higher absorbance means there are ?
MORE DNA
RATION OF A PURE DNA
1.8-2.0
RATIO WITH <1.8 MEANS
PROTEIN AND SOLVENT CONTAMINATION
RATION WITH >2.0 MEANS
rna contamination/ presence of chloroform or phenol
- Short single-stranded synthetic oligodinucleotide that
serve to start the DNA synthesis, which are
complementary to the ends of each target DNA.
primer
are synthetic oligonucleotides that serve to
initiate DNA synthesis.
primers
A pair of primers is required, called ??
forward and reverse
primers
- Primers range from ____ nucleotides and are single
stranded.
15-30
________ temperature refers to the DNA
molecule
o Melting
the temperature level at
which 50% of the DNA template are singlestranded and 50% are double stranded.
melting temperature
each primer should not have
complementary bases WITHIN the sequences to
prevent FORMATION OF ?
HAIRPIN LOOP.
- Each primer should not have complementary
sequences BETWEEN F primers and R primers will
FORMATION OF ?
PRIMER-DIMER.
means accurate synthesis.
high fidelity
If there are excess dNTPs, the _____________
will bind to it.
magnesium
- building blocks
for the new DNA.
Deoxynucleotide triphosphates (dNTPs)
The recommended concentration of each dNTP is ______
uM in each reaction mixture.
200-250
Optimum temperature is 72°C
o Makes an error in every 125, 000th nucleotides
o Typically, the recommended concentration in the
reaction mixture is 0.02-0.03 U/uL (unit per
microliter)
THERMUS AQUATICUS
o Has proofreading ability
o High fidelity (accuracy) DNA polymerase
o Makes error only in every 676, 000th nucleotid
Pfu DNA Polymerase
o Pyrococcus furiosus
o
o It controls the shift of pH by regulating the amount
or the concentration of hydrogen ions in the
solution.
Tris-HCl buffer
- Regulates amount of H+ ions in the solution
- Specific pH level: 8.0-9.5
PCR BUFFERS
important for DNA Polymerase. It serves
as a cofactor, which is important for the specificity of
annealing of primers.
MAGNESIUM
the enzyme of choice for routine
amplification of small segments of DNA. Its optimal
temperature at 72°C.
TAQ POLYMERASE
o If there’s too much hydrogen ion in the solution, the
hydrogen ion will bind to the phosphate group of
dNTPs leading to the formation of ?
phosphoric acid.
- Water used in the reaction mixture must be
?
nuclease-free.
a machine that automatically changes
the temperatures and incubation times in each cycle of
the PCR
THERMOCYCLER
- The ideal dNTP concentration is ____ µM of each, but
some enzymes may require as much as 400 µM each.
200
In dNTPs Lower concentration can _________ enzyme fidelity but the
yield may be reduced.
INCREASE
- A method of separating electrically charged substances
in a mixture (e.g., DNA, RNA, proteins, etc.).
electrophoresis
- A method of separating electrically charged substances
in a mixture (e.g., DNA, RNA, proteins, etc.).
electrophoresis
This electrophoresis technique was first developed by ? in 1930
for the study of serum proteins
Arne Tiselius
- A type of electrophoresis in which supporting medium
is a gel
GEL ELECTROPHORESIS
ELECTROPHORESIS: 3 PARTS are
voltage, supporting medium, buffer system
____________ gel structure held together by covalent
cross-linking of Acrylamide and N, N’-methylene
bisacrylamide
* Tougher than agarose gels
Polyacrylamide Gel (PAGE)
- Most effective in separating DNA fragments of
varying sizes from 100bp to 25kb.
what type of gel is this
agarose gel
- It is a linear carbohydrate polymer composed of
repeating units of agarobiose, which consists of
alternating units of galactose and 3,6-anhydrogalactose
agarose gel
- This is a procedure that separates molecules based on their rate of movement through agarose gel under the influence of an electrical field.
AGAROSE GEL ELECTROPHORESIS
- It is placed in the liquid agarose after it has been poured
- Removing the it from the hardened gel produces
a series of wells used to load the DNA.
comb
- Its function is to prevent the pH from changing. It is used
to resist pH changes.
running buffer
used in analyzing
large sizes (around 1500 bp or higher); generates
less current compared to TBE
tris acetate EDTA
used in analyzing
smaller sizes or fragments.
tris borate EDTA
- Also known as Molecular
Weight Marker - It is usually dispensed in
the first lane of the well.
DNA LADDER
most used DNA stains
ethidium bromide
oDerived from two species of seaweeds: Gelidium
and Glacilaria.
- STANDARD (HIGH-MELTING-TEMPERATURE)
AGAROSES
Gel casted horizontally in this gel
agarose
this gel is Commonly used for DNA
separations
agarose
Running buffer too diluted,
voltage too high results to
a poor resolution
- This is used to separate very large DNA molecules.
- This process uses more than one electrical field (e.g., one
direction, horizontal, vertical, or opposite direction).
pulse-field gel electrohporesis
: The mobility of DNA molecules is HIGHLY
DEPENDENT ON THE ELECTRICAL FIELD applied to
the gel, because it disrupts hydrogen bonds t or f
true !!
