MOLECULAR DIAGNOSTICS EXAM #2
QUESTIONS WITH CORRECT
ANSWERS
p. 110 nucleic acid specific dyes - SYBR green - ANSWER-SyBr green in
association with DNA or RNA also emits light in the orange range (522 nm). In
agarose gel electrophoresis, SyBr staining is 25-100 times more sensitive than EtBr
(detection level: 60 pg of double-stranded DNA vs. 5 ng for EtBr). This is due, in part,
to background fluorescence from EtBr in agarose.
SyBr green can also be added directly to the DNA sample before electrophoresis.
DNA prestaining decreases the amount of dye required for DNA visualization but
lowers the sensitivity of detection and may, at higher DNA concentrations, interfere
with DNA migration through the gel.
Because SyBr green is not an intercalating agent, it is not as mutagenic and is
therefore safer to use.
p. 117 Southern Blots - specificity and procedure steps - Digestion, Electrophoresis,
Denaturation, Transfer, Probe Hybridization) - ANSWER-In the Southern blot, DNA
is isolated and cut with restriction enzymes. The fragments are separated by gel
electrophoresis, denatured, and then transferred to a solid support such as
nitrocellulose.
In the final steps of the procedure, the DNA fragments are exposed to a labeled
probe (complementary DNA or RNA) that is complementary to the region of interest.
The signal of the probe is detected to indicate the presence or absence (lack of
signal) of the sequence in question.
p. 118 Depurination - how is it done (HCl) - ANSWER-Before moving the DNA
fragments from the gel to the membrane for blotting, the double stranded DNA
fragments are denatured as the DNA remains in place in the gel. Although short
fragments can be denatured directly, larger fragments (greater than 500 bp) are
more efficiently denatured if they are depurinated before denaturation.
Therefore, for larger fragments, the gel is first soaked in dilute hydrogen chloride
(HCl) solution, a process that removes purine bases from the sugar phosphate
backbone. This will loosen up the larger fragments for more complete denaturation.
p. 119 Membrane types what's used the most? Nitrocellulose - ANSWER-Single
stranded DNA avidly binds to nitrocellulose membranes with a noncovalent, but
irreversible connection.
Pure nitrocellulose has a high binding capacity for protein as well as nucleic acids. It
is the most versatile medium for molecular transfer applications. It is also compatible
with different transfer buffers and detection systems.
, p. 120-121 Transfer methods - name them - ANSWER-Capillary transfer = no
instruments needed, but the transfer is less than optimal and procedure is slow
taking hours to overnight.
Electrophoretic transfer = uses electric current to move DNA from the gel to the
membrane
Vacuum transfer = blotting technique uses suction to move the DNA from the gel to
the membrane in a recirculating buffer.
p. 122 Northern Blot - what does it measure? RNA, gene expression - ANSWER-The
northern blot technique was designed to investigate RNA structure and quantity.
Although most northern analyses were performed to investigate levels of gene
expression (transcription from DNA) and stability, the analyses were also used to
investigate RNA structural abnormalities resulting from aberrations in synthesis or
processing, such as alternative splicing.
p. 123 probes - what do they do? - ANSWER-The purpose of the probe is to identify
one or more sequences of interest within a large amount of nucleic acid.
The probe therefore should hybridize specifically with the target DNA or RNA that is
to be analyzed. The probe can be RNA, denatured DNA, or other modified nucleic
acids.
Probes for western blots are specific binding proteins or antibodies.
p. 128 melting temperature definition - ANSWER-The ideal hybridization conditions
are estimated from the calculation of the melting temperature or Tm of the probe
sequence. The Tm is a way to express the amount of energy required to separate
the hybridized strands of a given sequence. At the Tm, half of the sequence is
double stranded, and half is single stranded.
p. 133 Dot/Slot blots best suited for what? Qualitative analysis, comparing many
targets for mutational screening - ANSWER-Dot blots are useful for multiple
qualitative analyses where many targets are being compared, such as in mutational
screening.
p. 134-135 Macro and Micro Arrays, reverse dot blot - ANSWER-Reverse dot blot =
many different probes are immobilized on the membrane and the test sample is
labeled for hybridization with the immobilized probes. Immobilized probe is now
effectively the target, and the labeled specimen DNA, RNA, or protein is actually the
probe(s). Regardless of the designation, the general idea is that a known sequence
is immobilized at a known location on the blot and the amount of sample that
hybridizes to it is determined by the signal from the labeled sample.
Macroarrays are reverse dot blots of up to several thousand targets on nitrocellulose
membranes
p. 135 Microarray and the analysis of the human genome - ANSWER-Microarray =
tens of thousands of targets could be screened simultaneously in a very small area
by miniaturizing the deposition of droplets. Automated depositing systems (arrayers)
QUESTIONS WITH CORRECT
ANSWERS
p. 110 nucleic acid specific dyes - SYBR green - ANSWER-SyBr green in
association with DNA or RNA also emits light in the orange range (522 nm). In
agarose gel electrophoresis, SyBr staining is 25-100 times more sensitive than EtBr
(detection level: 60 pg of double-stranded DNA vs. 5 ng for EtBr). This is due, in part,
to background fluorescence from EtBr in agarose.
SyBr green can also be added directly to the DNA sample before electrophoresis.
DNA prestaining decreases the amount of dye required for DNA visualization but
lowers the sensitivity of detection and may, at higher DNA concentrations, interfere
with DNA migration through the gel.
Because SyBr green is not an intercalating agent, it is not as mutagenic and is
therefore safer to use.
p. 117 Southern Blots - specificity and procedure steps - Digestion, Electrophoresis,
Denaturation, Transfer, Probe Hybridization) - ANSWER-In the Southern blot, DNA
is isolated and cut with restriction enzymes. The fragments are separated by gel
electrophoresis, denatured, and then transferred to a solid support such as
nitrocellulose.
In the final steps of the procedure, the DNA fragments are exposed to a labeled
probe (complementary DNA or RNA) that is complementary to the region of interest.
The signal of the probe is detected to indicate the presence or absence (lack of
signal) of the sequence in question.
p. 118 Depurination - how is it done (HCl) - ANSWER-Before moving the DNA
fragments from the gel to the membrane for blotting, the double stranded DNA
fragments are denatured as the DNA remains in place in the gel. Although short
fragments can be denatured directly, larger fragments (greater than 500 bp) are
more efficiently denatured if they are depurinated before denaturation.
Therefore, for larger fragments, the gel is first soaked in dilute hydrogen chloride
(HCl) solution, a process that removes purine bases from the sugar phosphate
backbone. This will loosen up the larger fragments for more complete denaturation.
p. 119 Membrane types what's used the most? Nitrocellulose - ANSWER-Single
stranded DNA avidly binds to nitrocellulose membranes with a noncovalent, but
irreversible connection.
Pure nitrocellulose has a high binding capacity for protein as well as nucleic acids. It
is the most versatile medium for molecular transfer applications. It is also compatible
with different transfer buffers and detection systems.
, p. 120-121 Transfer methods - name them - ANSWER-Capillary transfer = no
instruments needed, but the transfer is less than optimal and procedure is slow
taking hours to overnight.
Electrophoretic transfer = uses electric current to move DNA from the gel to the
membrane
Vacuum transfer = blotting technique uses suction to move the DNA from the gel to
the membrane in a recirculating buffer.
p. 122 Northern Blot - what does it measure? RNA, gene expression - ANSWER-The
northern blot technique was designed to investigate RNA structure and quantity.
Although most northern analyses were performed to investigate levels of gene
expression (transcription from DNA) and stability, the analyses were also used to
investigate RNA structural abnormalities resulting from aberrations in synthesis or
processing, such as alternative splicing.
p. 123 probes - what do they do? - ANSWER-The purpose of the probe is to identify
one or more sequences of interest within a large amount of nucleic acid.
The probe therefore should hybridize specifically with the target DNA or RNA that is
to be analyzed. The probe can be RNA, denatured DNA, or other modified nucleic
acids.
Probes for western blots are specific binding proteins or antibodies.
p. 128 melting temperature definition - ANSWER-The ideal hybridization conditions
are estimated from the calculation of the melting temperature or Tm of the probe
sequence. The Tm is a way to express the amount of energy required to separate
the hybridized strands of a given sequence. At the Tm, half of the sequence is
double stranded, and half is single stranded.
p. 133 Dot/Slot blots best suited for what? Qualitative analysis, comparing many
targets for mutational screening - ANSWER-Dot blots are useful for multiple
qualitative analyses where many targets are being compared, such as in mutational
screening.
p. 134-135 Macro and Micro Arrays, reverse dot blot - ANSWER-Reverse dot blot =
many different probes are immobilized on the membrane and the test sample is
labeled for hybridization with the immobilized probes. Immobilized probe is now
effectively the target, and the labeled specimen DNA, RNA, or protein is actually the
probe(s). Regardless of the designation, the general idea is that a known sequence
is immobilized at a known location on the blot and the amount of sample that
hybridizes to it is determined by the signal from the labeled sample.
Macroarrays are reverse dot blots of up to several thousand targets on nitrocellulose
membranes
p. 135 Microarray and the analysis of the human genome - ANSWER-Microarray =
tens of thousands of targets could be screened simultaneously in a very small area
by miniaturizing the deposition of droplets. Automated depositing systems (arrayers)
