MIDTERM REVIEW Flashcards
Which of the following are FALSE about automated Sanger (dye terminator) DNA sequencing?
a) A different colour fluorescent dye is used to tag each type of ddNTP
b) The DNA primer used is unlabeled (no radioactive or fluorescent tag)
c) It is possible that more than one dideoxy nucleotide could be incorporated in the same DNA strand.
d)All 4 didexy reactions could be performed in the same tube and loaded into the same lane of the gel.
e)None of the statements are false
f) All statements are false
c)
What is FALSE about the DNA sequencing of genomes?
a) The first human genome sequenced was that of James Watson
b) the sequencing of the first human genome cost > 3 billion
c) Sanger sequencing was used to generate the first human genome
d) Although Maxam & Gilbert method was developed around the same time as the Sanger method, the Maxam & Gilbert method is rarely used for sequencing an entire genome.
e) Although 1000’s unique animals had their nuclear genomes sequenced the type of animals that had their genomes sequenced is greatly biased towards vertebrates.
a) or b)
Which of the following is FALSE about the shotgun sequencing strategy?
a) short fragments of genomic DNA are inserted into vectors such as plasmids
b) A genomic library is a collection of E. coli that are transformed by plasmids containing different fragments of genomic DNA
c) The DNA sequence of the inserted fragment in each plasmid is determined.
d) The DNA sequences are assembled into contigs
e) This method is costly since new primers are needed for sequencing each DNA fragment
f) none of the above statements are false
e)
Which of the following are FALSE about Illumina sequencing?
a) It is not necessary to fragment or cut the genomic DNA into smaller pieces
b) It is not necessary to insert the genomic DNA into a cloning vector such as a plasmid or a BAC
c) This method is fast because there is no interruption of DNA synthesis performed by the DNA polymerase
d) Flashes of light are recorded after each cycle of DNA sequencing for pyrosequencing and Illumina sequencing
e) All of the above are false
f) None of the above is false
a) or c)
Which of the following is/are FALSE about Next Generation or 2nd Gen sequencing technologies?
a) They allow many more nucleotides sequences to be determined per day than the Sanger method
b) They can obtain much more accurate nucleotide sequences than the Sanger method
c) They can sequence complete genomes at much lower costs than the Sanger method
d) Pyrosequencing is one type of Next Gen sequencing
e) All of the above are false
f) None of the above are false
b)
Fill in the blanks of the following statement: the fluorescent tag in Illumina sequencing is attached to _______, and in PacBio sequencing the tag is attached to _______ of the nucleotide.
a) The 5’ phosphate group, 2’ carbon of the sugar
b) The base, 3’carbon of the sugar
c) The 2’ carbon of the sugar, 3’carbon of the sugar
d) 2’ carbon of the sugar, the base
e) The base, 5’ phosphate group
e)
What causes DNA synthesis to stop in the Illumina method of DNA sequencing?
a) release of a pyrophosphate by the nucleotide
b) Inactivation of the DNA polymerase
c) Incorporation of a modified nucleotide
d) Depletion of nucleotides in the reaction
e) The DNA polymerase reaches the end of the DNA template
c)
Fill in the blanks of the following statement: _______ is a type of RNA responsible for blocking translation of specific mRNAs, and ______ is responsible for regulating the movement of transposons in the DNA of germ line cells.
a) siRNA, tRNA
b) miRNA, piRNA
c) snRNA, miRNA
d) InRNA, snoRNA
e) rRNA, tRNA
b)
A Partial sequence of an RNA is shown below. Which of the following DNA strands represent the TEMPLATE strand of the corresponding DNA?
a) 5’…GTACATTAC…3’
b) 5’…CATGTAATG…3’
c) 5’…CATTAGATC…3’
d) 5’…GTAATGTAC…3’
e) None of the above
d)
Which of the following is FALSE about reading frames?
a) There is a maximum of 3 possible reading frames for each single strand of DNA.
b) The reading frames are based on the specific grouping of three DNA nucleotides to form a triplet codon.
c) A stop codon is expected to occur once in every 21 codons by chance
d) The correct reading frame for a gene could be in either the top strand of the bottom strand of a region of double-stranded DNA
e) Once the reading frame is established for gene X, genes located immediately upstream or downstream of gene X will also have the same reading frame.
e)
How is the primer walking method of DNA sequencing performed?
(short answer)
- use a primer that is complimentary to a known sequence at the start of DNA template.
- Sequence the DNA template using the initial primer.
- Identify the newly obtained sequence data from the sequencing reaction.
- Create a new primer that binds to the end of the newly sequenced region.
- Chemically synthesize the new primer.
- Use the new primer for another round of DNA sequencing to extend the sequence.
Contig assembly for DNA sequence data obtained from “primer walking” is easier to do than data obtained from “shotgun sequencing” strategy. Explain why this is in no more than 1-2 sentences.
Contig assembly for DNA sequence data obtained from primer walking is easier because the fragments are already ordered sequentially, as primers are designed step-by-step based on the known sequence. This eliminates the need for computational reassembly like in shotgun sequencing.
Why is it unnecessary to incorporate a PCR step to increase the amount of genomic DNA as templates for PacBio sequencing?
PCR is not necessary for PacBio sequencing because the technology is sensitive enough to directly sequence single molecules of DNA without needing amplification.
Briefly describe how DNA sequencing is performed by the MinION method.
DNA sequencing by the MinION method involves passing a single-stranded DNA molecule through a nanopore, where changes in ionic current as each nucleotide passes through are measured. These current changes are then translated into the DNA sequence in real-time.
What is the main advantage of Illumina sequencing over Sanger sequencing?
a. Ability to sequence longer fragments
b. Higher throughput and cost efficiency
c. Higher accuracy per base
d. Does not require PCR amplification
Answer: b. Higher throughput and cost efficiency
