Molecular Biology Week 3 Flashcards
Name three types of enzymes that are used in a molecular biology lab?
Polymerase, Nucleases, Ligase
What do Polymerases do?
Polymerases create polymers of nucleic acids ex: RNA and DNA
How do polymerases
make new DNA or RNA?
New DNA/RNA is made using an existing DNA or RNA as a template of info for bp rules.
Give an example for each DNA and RNA for a polymerase in vivo.
DNA ; rep and rep
RNA: transcription
What are nucleases capable of doing?
They are capable of cleaving phosphodiester bonds between nucleotide (phos, sug and b). They can be both endonucleases and exonucleases (5’-3’ or 3’ to 5’)
What is an example of a nucleases in vivo?
DNA rep and apoptosis
What do DNA ligases do?
Ligases join DNA strands together by being a catalyst in the formation of a phosphodiester bond.
Give an example of a ligases in vitro.
Cloning
What is the biological function of restriction endonucleases in the bacteria that produce them?
Is to recognise one or more restriction sites and to make a double stranded cut in the DNA molecule. This cut occurs at the restriction site and occurs in an orderly pattern.
What are the two different types of cuts made by RE and distingush between the two of them.
Pvull, allul are examples of blunt/protruding ends this involves the cleaving of both strands at SAME point of DNA. Uniform edges with NO overhangs.
Cutting of two strands of DNA at differnt points are called staggered cuts, this results in the generaton of blunt ends these are also known as staggered or sticky ends with complimentary overhangs.
What does a restriction site look like and what is it called with the mirrored bp?
5’ - G - A - A- T - T - C - 3’ Palindromes
What is the importance of the single stranded overhang created in some RE particularly useful?
Produces highly reactive sticky ends on the brokwn ends which are helpful for genetic engineering and cloning as these ends can stick together for long enough for the enzyme ligase to bind the two strands together.
How are RE named?
Strain, Haemophilius Influenzae = HIND III
First three letters - abbreviate genus(H) and species (IN)
Fourth letter - comes from the bacterial strain designation (D)
Roman numerals indicates the order in which restriction enzymes were discovered in a particular strain (I, II, III)
What is the difference between an RE and an exonuclease?
Exonucleases remove nucleotides from the ends of a nucleic acids molecule 5’ or 3’ end.
An exonuclease never produces internal cuts in the DNA
What are the two forms of DNA replication?
Mitosis, Binary fission (bacteria)
Since DNA double helix strands are complementary what does this mean in terms of the process of DNA replication?
These strands can be the template for DNA replication, the process is semi-conservative asa each daughter duplex has one parental strand and one new strand
What are the two other theories proposed about DNA replication other than that of being semi-conservative.
Can either be fully conservative or dispersive
Whos experiments proved that the process was definitely semi-conservative?
Meselson-Stahl
Describe the three postulated models of DNA replication.
Conservative, both old,
Semi-conservative, new and old.
Dispersive, old + new parts added.
Describe the Meselson Stahl Experiment.
They grew E.coli in heavy nitrogen 15 , gre also in nitrogen 14. Mixed together. First gen (time 0) all heavy 100%, next gen (20 min) 100% hybrid), after that the samples were half hybrid half light. No heavy DNA. Showed that DNA replication uses templates, splits, reads, adds new complementary strand.
What direction does DNA polymerase catalyse in and starts at which part?
5’ to 3’ direction, DNA polymerase can only add a nucleotide to a free 3’ OH.
Is DNA polymerase template dependent?
Yes it is, it reads the sequence of bases on the template strands and inserts the appropriate complementary base into the new strand.
What are the sites in the DNA knows as where the replication begins?
Origins of Replication
What enzyme must proceed first to initiate the process of replication? What shape does this produce in the DNA
Helicase enzymes must unwind the double helix structure of the DNA. gradually This produces a replication fork.
What did Okazaki deduce?
That while one strand is being synthesised, the fork opening this is called the leading strand, the other is made discontinuously as short segments of DNA in the 5’ to 3’ direction are joined together to make a new strand called the lagging strand. The short fragments being joined onto this strand are called Okazaki fragments.
DNA replication is ——– discontinuous.
self-discontinuous.
What is the primer enzyme that is used in the initiation of DNA synthesis?
Primase enzyme to the 3’ OH which nucleotides are added to, these are the okazaki fragments that are then subsequently are plugged in
What removes the primase enzyme and fills in the gaps?
Exonucleases remove the primers, DNA ligase then joins the 2 strands together. DNA polymerase fills the gaps.
