first genome project
The year of the first genome project
What is 1995?
How do restriction enzymes act as a defense mechanisms and what are they defending against?
They cut foreign viral DNA
What are key characteristics of Yeast Artificial Chromosomes?
Limit of 100-1000 Kb
Linear chromosomes that contain: centromeres, telomeres, origin of replication, restriction enzyme recognition site, and amp R site
What are the starting materials of PCR?
Template DNA, synthesized primers, and an abundant amount of all 4 dNTP's
It does not have an OH at the 3' carbon so it halts elongation
The name of the virus that was studied and its effect
What is Haemophilus influenza that kills babies ages 4-18 months??
How do restriction enzymes select a DNA strand? What is unique about these?
They have a unique recognition site in the DNA sequence to which they bind.
Each recognition site is a palindrome - it can be read the same forwards and backwards.
What are key characteristics of Bacterial Artificial Chromosomes?
Limit: 75-300 Kb
Circular synthesized chromosome that contains: origin of replication, AmpR gene, multiple cloning site, and desired genes
What is the first step of PCR called and what occurs during it?
Denaturation - the tube is heated to 95 degrees C to denature/separate the double stranded template DNA
How many tubes are used to conduct Sanger Sequencing and why?
Four tubes, one for each nucleotide
The number of base pairs in the virus' genome
What is 2,000 Kb
How does the bacteria protect its own genome from being cut by the restriction enzyme?
They methylate their A and C bases in their genome.
What is a dNTP? (What does it stand for and what are its components?)
Deoxyribonucleotide triphosphate
Contains a phosphate group, a deoxyribose sugar and a nitrogenous base
What is the second step of PCR called and what occurs during it?
Primer Annealing - cool to 50-60 degrees C so that primers bond to the template DNA with complementary base pairing
How are the identities of the final nucleotide identified in the synthesized DNA fragments?
They are fluorescently labeled, each with a different color, and a laser is used to see their colors.
The method used to sequence the entire virus genome
What is Shotgun sequencing?
Depending on the type of cut (zig-zag versus straight), the resulting DNA can have two types of ends. What are they?
Sticky ends and blunt ends.
What is the significance of the Amp R gene?
It allows only the cells which took in the desired plasmid to live and replicate in an ampicillin-rich environment, therefore reproducing the plasmid and producing the desired product
What is the third step of PCR called and what occurs during it?
Extension - heat to 68 degrees C so that the polymerase synthesizes the complementary DNA using the dNTPs
What is a key assumption of Sanger sequencing using diNTP's?
We can create a fragment of every single length if we run the PCR-like reaction with the diNTP's for long enough
How does shotgun sequencing work? (4 distinct "steps")
1. It cute the whole genome into fragments
2. The fragments are cloned into plasmids
3. The end of each fragment is sequenced
4. Fragments with the same ends are overlapped to form one long contiguous sequence
What specific bond is cut by the restriction enzyme?
Phosphodiester bond of the DNA backbone
What is a famous, popular example of the use of restriction enzymes in modern-day science? Briefly explain how it works.
Insulin Production
What is the specific type of DNA Polymerase used in PCR and from where did it come?
Taq polymerase which came from an archaea extremophile so that it can be used in PCR and survive the 95 degree C heating
How are the fragments separated/ordered by length?