Lecture 5 - DNA sequencing and the Polymerase Chain Reaction Flashcards
What are the two essential features for DNA synthesis by DNA polymerase?
- a short segment of double-stranded nucleic acid serves as a primer for initiation of DNA synthesis
- DNA is synthesized on the 5’ to 3’ direction
Both DNA sequencing and the polymerase chain reaction requires what?
DNA synthesis in vitro
What are the minimum requirements for DNA synthesis in vitro?
a DNA template, DNA polymerase, DNA primer and Deoxyribonucleoside triphosphates
What was the method created by Maxam and Gilbert, that was published in 1977 for DNA sequencing and why is it no commonly used now?
The chemical cleavage method, not commonly used now because it could lead to cancer.
what method did Fred Sanger publish in 1975 that is a very popular method and now semi-automated?
Enzymatic dideoxy chain terminating method
What did Sanders recognize that can terminate the synthesis of DNA?
ddNTP because it does not contain the second oxygen required for DNA synthesis
What is the strategy for DNA sequencing by the dideoxy chain termination method?
- we run same reactions in different tubes to determine different positions, and when run on gel, the protein is denatured and the primer is radioactive
- you will then be able to detect the primer on an x-ray film
What direction is the DNA sequence read on the gel?
From the bottom of the gel to the top in the 5’ to 3’ direction. The smaller DNA fragments migrate more rapidly.
Which sequence is read on the gel?
The synthesized strand, which is complementary to the template DNA strand
Based on what, can we determine how the DNA molecule will fold up?
Based on the amino acids and the protein composition
What are some consequences of genome sequencing?
- the genetic relationship of species
- genome evolution
- medical genetics
- explains how chromosomes have evolved and changed
Where did humans originate from?
East Africa ( maybe 100,000 or 70,000 years ago
Who invented the Polymerase Chain Reaction (PCR) for amplification of targeted DNA fragments?
Dr. Kary Mullis
What did Kary Millis recognize with his invention?
That you can take a very small amount of DNA and amplify it many times, if you understand what the requirements are (promoter, etc…)
Automate PCR with a computer-driven thermal cycler, and Taq polymerase, a DNA polymerase from the bacterium, Thermus aquaticus, does not denature at high temperatures are requirements for what?
Efficient PCR
