Quiz 2- Lecture 7: DNA Sequencing Flashcards
(40 cards)
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”
