Quiz 2- Lecture 7: DNA Sequencing Flashcards
What does DNA Sequencing refer to?
What do the sequence of the bases encode?
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
What is epigenetics?
… with environment
Do you have to know both strands?
No, the strands are complimentary so if you know one side, you can figure out the other
What were Robert Holley and his colleagues the first to do?
Sequence yeast transfer RNA (tRNA) using RNAses with base specificity in 1965 (alanine tRNA from Saccharomyces cerevisiae)
What was established in the 1950s?
Genetic information is transferred from DNA to RNA, to protein
What is a codon?
What does it do?
A sequence of three nucleotides in DNA
Corresponds to a particular amino acid in a protein
What is a ribosome?
What is tRNA
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
Who was Robert Holley?
The first person to successfully isolate tRNA and, in 1964, was also able to map its structure
What are the 3 types of RNA?
Messenger, Transfer, Ribosomal
What does tRNA do?
What are its 2 hands?
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
Separation techniques: Chromatography
What is Chromatography?
An important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis
What is Counter-current distribution (also known as liquid-liquid extraction or partition chromatography)?
What does it involve?
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
Countercurrent Disrribution:
What can sequential countercurrent extractions do?
Sequential countercurrent extractions can separate solutes with only small differences in K
(However, the technique, if performed with separatory funnels, is quite tedious
Who was Lyman C. Craig?
In 1944 Lyman C. Craig developed a device to automate countercurrent distribution
This device used a series of glass vessels
Transfer RNA:
What does Figure 4 show? (4)
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
Transfer RNA:
What does figure 12 show?
Suggested secondary structure of the alanine transfer RNA
Transfer RNA:
What did I write?
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
tRNA:
What do the figures show? (2)
(a) The cloverleaf structure of tRNA with its bases numbered
(b) Schematic diagram of the three-dimensional structure of yeast tRNA^Phe
In 1972, Walter Fiers was first to…
What did he utilize? To do what? (2)
How did he separate them?
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
H. Gobind Khorana
Nobel Prize in Physiology or Medicine 1968
Prize motivation: “for their interpretation of the genetic code and its function in protein synthesis”
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?
An alternative DNA sequencing method and in 1977, developed the first DNA sequencing method that utilized Radiolabeled partially digested fragments called
“Chain termination method”
What method went on to dominate the sequencing world for the next 30 years?
What is Sanger considered as?
Chain termination method
A giant in genomics
DNA to be sequenced… (3 figures)
What does figure b show?
(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)
Paul Berg
Walter Gilbert
Frederick Sanger
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”
What is Maxam and Gilbert’s Chemical Sequencing Method?
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
Hydrazine removes bases from what? (2)
Acid removes bases from what? (2)
Piperidine is used to do what?
Fragments can do what?
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
What is the sequencing of 5’ to 3’ DNA mean?
5’ means Phosphate group
3’ means Hydroxil group
What does DNA denaturation mean?
The strands will separate
What does DNA radioactive labeling mean?
Adding radioactive (isotope of) phosphate
What is Purine?
Type of Nitrogen Base (4 different nitrogen bases)
Why is this method (second-generation DNA sequencing) markedly differed from existing methods?
Instead, what did researchers utilize?
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
What was this approach used to infer?
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
Despite the difference, Sanger’s dideoxy and this pyrosequencing are both what?
How?
“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)
What does the PCR primer figure show?
-> 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
What does Sulfurylase figure show?
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)
What does the figure about dNTP and ATP show? (2)
dNTP -(Apyrase)-> dNDP + dNMP + phosphate
ATP -(Apyrase)-> ADP + AMP + phosphate
What does Nucleotide sequence - Nucleotide added figure show?
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
What does Third generation slide say?
Third-generation DNA sequencing nucleotide detection
What does DNA microbead figure show? (4)
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)”
What does Fluorescent dNTPs figure show? (2)
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)
