DGS 4234 EXAM 3 QUESTIONS WITH ANSWERS
100% PASS
allows us to find the precise order of nucleotide bases in a DNA fragment - ANSWER
what does DNA sequencing accomplish?
1. Maxam-Gilbert sequencing
2. relied on radioactively labeled DNA
3. efficiently determined short runs of sequence data
4. not practical for high throughout sequencing of long fragments
5. hazardous chemicals were used - ANSWER describe chemical sequencing
1. chain termination chemistry
2. "gold standard" for mutation detection
3. historically, used high-resolution denaturing polyacrylamide gels
4. now used with capillary electrophoresis
5. resolves fragments of ssDNA that differ by a single base - ANSWER describe Sanger
sequencing
1. dideoxynucleotide
2. it has no -OH so no further nucleotides can be added and chain cannot be extended -
ANSWER what doe Sanger sequencing incorporate into the DNA chain to stop extension
and why does this molecule successfully terminate extension?
1. DNA is fragmented
2. DNA is cloned to a plasmid vector for amplification in vivo OR PCR amplification in
vitro
3. cyclic sequencing (chain termination reaction)
,4. separation by electrophoresis
5. detection of fluorescent labels
6. interpretations (many artifacts can occur so it's important to know what you're
looking at) - ANSWER what are the steps in Sanger sequencing?
1. mutation detection
2. confirmation of mutation detection by other methods
3. resistance testing (drug resistance)
4. HLA genotyping (transplantation) - ANSWER what are some clinical applications of
DNA sequencing?
1. PCR amplification of target DNA region
2. cleaning of PCR product to get rid of excess primers, dNTPs (using spin columns, etc)
- ANSWER what happens before the sequencing reaction?
1. PCR amplification and cleaning of product
2. sequencing reaction (incorporating label)
3. removal of excess reagents (label)
4. detection of labeled fragments (capillary electrophoresis)
5. finishing (assembling raw sequence reads into an accurate, contagious sequence) -
ANSWER what the basic process of sequencing?
the 5' end - ANSWER where are universal primers added to?
1. with dye-labeled sequencing primers
2. with dye terminator chemistries
3. to simplify and standardize sequencing step - ANSWER when are universal-tailed PCR
primers used?
, 1. primers should be specific for the target sequence and should not have any
secondary structures
2. should not have any repeating motifs
3. primer pairs should have compatible melting temperature
4. 50% GC content
5. 3' end rich in GCs to enhance annealing of end that will be extended
6. should not have complementarity in order to prevent primer dimers - ANSWER what
are important aspects of having an optimal primer design?
to confirm mutations or polymorphisms - ANSWER why are both complementary strands
of DNA sequenced in Sanger sequencing?
1. excess dye terminator removed
2. spin columns/bead systems bind the sequencing fragments to remove residual
sequencing components by rinsing with buffer
3. sequence fragments denatured - ANSWER how are sequence fragments prepared for
fluorescence detection?
so that the ss fragments are resolved strictly according to size - ANSWER why are the
denaturing conditions maintained when preparing sequence fragments for fluorescence
detection?
1. 50-60C
2. formamide - ANSWER what are the denaturing conditions for sequence fragment
prep?
label the fragments synthesized during the sequencing reaction according to their
terminal ddNTP - ANSWER what is the goal of automated fluorescent sequencing?
fragments labeled with fluorescent dyes that have distinct peak wavelengths of
emission that can be distinguished by the automated sequencers (either dye primer
100% PASS
allows us to find the precise order of nucleotide bases in a DNA fragment - ANSWER
what does DNA sequencing accomplish?
1. Maxam-Gilbert sequencing
2. relied on radioactively labeled DNA
3. efficiently determined short runs of sequence data
4. not practical for high throughout sequencing of long fragments
5. hazardous chemicals were used - ANSWER describe chemical sequencing
1. chain termination chemistry
2. "gold standard" for mutation detection
3. historically, used high-resolution denaturing polyacrylamide gels
4. now used with capillary electrophoresis
5. resolves fragments of ssDNA that differ by a single base - ANSWER describe Sanger
sequencing
1. dideoxynucleotide
2. it has no -OH so no further nucleotides can be added and chain cannot be extended -
ANSWER what doe Sanger sequencing incorporate into the DNA chain to stop extension
and why does this molecule successfully terminate extension?
1. DNA is fragmented
2. DNA is cloned to a plasmid vector for amplification in vivo OR PCR amplification in
vitro
3. cyclic sequencing (chain termination reaction)
,4. separation by electrophoresis
5. detection of fluorescent labels
6. interpretations (many artifacts can occur so it's important to know what you're
looking at) - ANSWER what are the steps in Sanger sequencing?
1. mutation detection
2. confirmation of mutation detection by other methods
3. resistance testing (drug resistance)
4. HLA genotyping (transplantation) - ANSWER what are some clinical applications of
DNA sequencing?
1. PCR amplification of target DNA region
2. cleaning of PCR product to get rid of excess primers, dNTPs (using spin columns, etc)
- ANSWER what happens before the sequencing reaction?
1. PCR amplification and cleaning of product
2. sequencing reaction (incorporating label)
3. removal of excess reagents (label)
4. detection of labeled fragments (capillary electrophoresis)
5. finishing (assembling raw sequence reads into an accurate, contagious sequence) -
ANSWER what the basic process of sequencing?
the 5' end - ANSWER where are universal primers added to?
1. with dye-labeled sequencing primers
2. with dye terminator chemistries
3. to simplify and standardize sequencing step - ANSWER when are universal-tailed PCR
primers used?
, 1. primers should be specific for the target sequence and should not have any
secondary structures
2. should not have any repeating motifs
3. primer pairs should have compatible melting temperature
4. 50% GC content
5. 3' end rich in GCs to enhance annealing of end that will be extended
6. should not have complementarity in order to prevent primer dimers - ANSWER what
are important aspects of having an optimal primer design?
to confirm mutations or polymorphisms - ANSWER why are both complementary strands
of DNA sequenced in Sanger sequencing?
1. excess dye terminator removed
2. spin columns/bead systems bind the sequencing fragments to remove residual
sequencing components by rinsing with buffer
3. sequence fragments denatured - ANSWER how are sequence fragments prepared for
fluorescence detection?
so that the ss fragments are resolved strictly according to size - ANSWER why are the
denaturing conditions maintained when preparing sequence fragments for fluorescence
detection?
1. 50-60C
2. formamide - ANSWER what are the denaturing conditions for sequence fragment
prep?
label the fragments synthesized during the sequencing reaction according to their
terminal ddNTP - ANSWER what is the goal of automated fluorescent sequencing?
fragments labeled with fluorescent dyes that have distinct peak wavelengths of
emission that can be distinguished by the automated sequencers (either dye primer
