Quiz 2- Lecture 7: DNA Sequencing

Question 1

What does DNA Sequencing refer to?

What do the sequence of the bases encode?

Answer 1

The general laboratory technique for Determining the exact sequence of nucleotides, or bases, in a DNA molecule

The sequence of the bases (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and operate

Question 2

What is epigenetics?

Answer 2

… with environment

Question 3

Do you have to know both strands?

Answer 3

No, the strands are complimentary so if you know one side, you can figure out the other

Question 4

What were Robert Holley and his colleagues the first to do?

Answer 4

Sequence yeast transfer RNA (tRNA) using RNAses with base specificity in 1965 (alanine tRNA from Saccharomyces cerevisiae)

Question 5

What was established in the 1950s?

Answer 5

Genetic information is transferred from DNA to RNA, to protein

Question 6

What is a codon?
What does it do?

Answer 6

A sequence of three nucleotides in DNA
Corresponds to a particular amino acid in a protein

Question 7

What is a ribosome?

What is tRNA

Answer 7

What the proteins are formed in, which lie outside the cell nucleus

Transfer RNA; the transportation of amino acids to these ribosomes take place with the help of this particular kind of RNA
There exists a special tRNA molecule for each codon

Question 8

Who was Robert Holley?

Answer 8

The first person to successfully isolate tRNA and, in 1964, was also able to map its structure

Question 9

What are the 3 types of RNA?

Answer 9

Messenger, Transfer, Ribosomal

Question 10

What does tRNA do?
What are its 2 hands?

Answer 10

tRNAs bring their amino acids to the mRNA in a specific order
Codon, Anticodon
This order is determined by the attraction between a codon, a sequence of three nucleotides on the mRNA, and a complementary nucleotide triplet on the tRNA, called an anticodon

Question 11

Separation techniques: Chromatography
What is Chromatography?

Answer 11

An important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis

Question 12

What is Counter-current distribution (also known as liquid-liquid extraction or partition chromatography)?

What does it involve?

Answer 12

Technique used in chemistry to separate and purify components of a mixture

It involves the repeated distribution or partitioning of solutes between two immiscible liquid phases that flow in opposite directions

Question 13

Countercurrent Disrribution:
What can sequential countercurrent extractions do?

Answer 13

Sequential countercurrent extractions can separate solutes with only small differences in K
(However, the technique, if performed with separatory funnels, is quite tedious

Question 14

Who was Lyman C. Craig?

Answer 14

In 1944 Lyman C. Craig developed a device to automate countercurrent distribution
This device used a series of glass vessels

Question 15

Transfer RNA:
What does Figure 4 show? (4)

Answer 15

Separation of ribonuclease TI digest fragments of alanine transfer RNA by chromatography on DEAE-cellulose

A-U-U-C-C-G (partial digestion with snake venom) -> (phosphodiesterase)
A-U-U-C-C
A-U-U-C +
A-U-U +
A-U +
A + mononucleotides

Fragments obtained by complete digestion of alanine RNA with takadiastase ribonuclease TI

Fragments obtained by complete digestion of alanine RNA with pancreatic ribonuclease

Question 16

Transfer RNA:
What does figure 12 show?

Answer 16

Suggested secondary structure of the alanine transfer RNA

Question 17

Transfer RNA:
What did I write?

Answer 17

Analyzed pieces and … constructed transfer RNA sequence

R: RNA was repeated and purified and used enzymes to chop RNA into different pieces used to identify its structure

Question 18

tRNA:
What do the figures show? (2)

Answer 18

(a) The cloverleaf structure of tRNA with its bases numbered
(b) Schematic diagram of the three-dimensional structure of yeast tRNA^Phe

Question 19

In 1972, Walter Fiers was first to…
What did he utilize? To do what? (2)
How did he separate them?

Answer 19

Sequence the DNA of a complete gene (the gene encoding the coat protein of the bacteriophage MS2
By utilizing RNAses to Digest the virus RNA and Isolate oligonucleotides,
And then separating them via Electrophoresis/Chromatography

Question 20

H. Gobind Khorana

Answer 20

Nobel Prize in Physiology or Medicine 1968
Prize motivation: “for their interpretation of the genetic code and its function in protein synthesis”

Question 21

The breakthrough in DNA sequencing: The first generation
In parallel to Fiers’ achievement, Fredrick Sanger kept working on what?
What did he develop? What did it utilize?

Answer 21

An alternative DNA sequencing method and in 1977, developed the first DNA sequencing method that utilized Radiolabeled partially digested fragments called
“Chain termination method”

Question 22

What method went on to dominate the sequencing world for the next 30 years?
What is Sanger considered as?

Answer 22

Chain termination method
A giant in genomics

Question 23

DNA to be sequenced… (3 figures)

What does figure b show?

Answer 23

(a) is illustrated undergoing Sanger sequencing (b)
Sanger’s “chain-termination” sequencing

Radio- or fluorescently-labelled ddNTP nucleotides of a given type- which once incorporated, prevent further extension- are included in DNA polymerization reactions at low concentrations (primed off a 5’ sequence, not shown)

Therefore, in each of the four reactions, sequence fragments are generated with 3’ truncations as a ddNTP is randomly incorporated at a particular instance of that base (underlined 3’ terminal characters)

Question 24

Paul Berg
Walter Gilbert
Frederick Sanger

Answer 24

The Nobel Prize in Chemistry 1980 was divided, one half awarded to Paul Berg “for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA”, the other half jointly to Walter Gilbert and Frederick Sanger “for their contributions concerning the determination of base sequences in nucleic acids”

Question 25

What is Maxam and Gilbert’s Chemical Sequencing Method?

Answer 25

DNA must first be labeled, typically by inclusion of radioactive P^32 in its 5’ phosphate moiety (shown here by P)
Different chemical treatments are then used to selectively remove the base from a small proportion of DNA sites

Question 26

Hydrazine removes bases from what? (2)

Acid removes bases from what? (2)

Piperidine is used to do what?

Fragments can do what?

Answer 26

Pyrimidines (Cytosine and Thymine)
While hydrazine in the presence of high salt concentrations can only remove those from cytosine

Acid can then be used to remove the bases from Purines (Adenine and Guanine), with dimethyl sulfate being used to attack guanines (although adenine will also be affected to a much lesser extent)

Piperidine is then used to cleave the phophodiester backbone at the abasic site, yielding fragments of variable length

Fragments generated from either methodology can then be visualized via electrophoresis on a high-resolution polyacrylamide gel: Sequences are then inferred by reading “up” the gel, as the shorter DNA fragments migrate fastest

Question 27

What is the sequencing of 5’ to 3’ DNA mean?

Answer 27

5’ means Phosphate group
3’ means Hydroxil group

Question 28

What does DNA denaturation mean?

Answer 28

The strands will separate

Question 29

What does DNA radioactive labeling mean?

Answer 29

Adding radioactive (isotope of) phosphate

Question 30

What is Purine?

Answer 30

Type of Nitrogen Base (4 different nitrogen bases)

Question 31

Why is this method (second-generation DNA sequencing) markedly differed from existing methods?

Instead, what did researchers utilize?

Answer 31

Did not infer nucleotide identity through using radio- or fluorescently-labeled dNTPs or oligonucleotides before visualizing with electrophoresis

Luminescent method for measuring pyrophosphate synthesis: this consisted of a two-enzyme process in which ATP sulfurylase is used to convert pyrophosphate into ATP, which is then used as the substrate for luciferase, thus producing light proportional to the amount of pyrophosphate

Question 32

What was this approach used to infer?

Answer 32

Infer sequence by measuring pyrohosphate production as each nucleotide is washed through the system in turn over the template DNA affixed to the solid phase

Question 33

Despite the difference, Sanger’s dideoxy and this pyrosequencing are both what?
How?

Answer 33

“Sequence-by-synthesis” (SBS) techniques
They both require the direct action of DNA polymerase molecules to produce the observable output (in contrast to the Maxam-Gilbert technique)

Question 34

What does the PCR primer figure show?

Answer 34

-> Forward PCR Primer
Biotinylated reverse PCR primer <-
(Pointing down) Biotinylated single-stranded template

Polymerase
3’ C G T C C G G A G G C C A A G T T C C A 5’

5’ G C A G GC C T 3’

Polymerase
(DNA)n + dNTP —> (DNA) n+1 + PPi

Question 35

What does Sulfurylase figure show?

Answer 35

Sulfurylase
(v pointing from here to)
APS + PPi ATP

luciferin oxyluciferin
(pointing from here to)
Luciferase
(v pointing from here to)
ATP Light

Nucleotide incorporation generations light seen as a peak in the Pyrogram trace
Points to graph (Light)L(Time)

Question 36

What does the figure about dNTP and ATP show? (2)

Answer 36

dNTP -(Apyrase)-> dNDP + dNMP + phosphate

ATP -(Apyrase)-> ADP + AMP + phosphate

Question 37

What does Nucleotide sequence - Nucleotide added figure show?

Answer 37

Nucleotide sequence
G C - A GG CC T
(arrow) —>

m. m. 0 m. t. t. t. m.
G C T A G C T
(arrow) —>
Nucleotide added

Question 38

What does Third generation slide say?

Answer 38

Third-generation DNA sequencing nucleotide detection

Question 39

What does DNA microbead figure show? (4)

Answer 39

a) Attached DNA, Microbead -(emPCR)-> Clonally amplified- bead bound libraries
Aqueous droplets in an oil emulsion

Bead -(emPCR)-> Clonally amplified- bead bound libraries

b) Bridge amplification - Flowcell-bound cluster

c) Picotitre walls containing DNA-bearing microbeads

d) Flowcell bound clusters

“Barcode”
“Bead (agarose)”

Question 40

What does Fluorescent dNTPs figure show? (2)

Answer 40

a) Fluorescent dNTPS (side) <100nm (top middle)
Excitation (-600 nm) (arrow pointing up- from bottom) Fluorescence (arrow pointing down)

b) Direction of current/translocation (pointing down)