Overview/intro

Question 1

What are the bases in DNA

Answer 1

A,C,G,T

Question 2

What are the bases in RNA

Answer 2

A,C,G,U

Question 3

What molecules are involved in storage, transfer and processing in the central dogma of biology

Answer 3

Storage: DNA
Transfer: RNA
Processing: Protein

Question 4

What makes DNA good at information storage

Answer 4

chemically stable, structure (mostly) independent of sequence, and easy to faithfully replicate.

It’s good for information storage because of the chemical structure of the bases (represented by A,T, C,
and G) and how they will only pair with each other in certain ways; A with T and C with G. Each pair is a rung on the double helix’s spiral Staircase. The two strands can be separated and copied independently. DNA is better than RNA for infor­mation storage as it is a more stable molecule, as since the
2’ -hydroxyl group from RNA being removed makes It’s backbone
Less susceptible to cleavage by hydrolysis.

Question 5

What makes RNA good at its job of transfer

Answer 5

short lived so suitable for transient expression.

Question 6

What makes proteins good at their job of processing

Answer 6

complex three-dimensional structure allows for the performance of a wide variety of chemical and structural tasks.

Question 7

Why functions would RNA need to be the sole precursor to life and why would it have been appropriate?

Answer 7

At a minimum, an RNA precursor of life must be able to self-replicate. This implies roles in both data storage and catalysis.
Data storage and retrieval:
• Watson-Crick base pairing.
• Chemically stable. Catalysis:
• To reduce the activation energy of metabolic reactions RNA would have to be able to bind substrates and reaction intermediates specifically.

Question 8

What are the 3 different configurations in which DNA could conceivably replicate? And which was it?

Answer 8

Dispersive, conservative or semi-conservative

It was found to be seem-conservative with the two complimentary strands of the helix separating during replication.
each serving as a template for the construction of a new, matching strand

Question 9

How could you confirm experimentally what mode of replication happens in DNA

Answer 9

You could grow cells in 15N enriched media till they have incorporated 15N into their DNA. These cells could then be transferred to 14N media. If one daughter received only copied DNA then a 15N band would persist while a new band at 14N would arise. On the other hand, if each daughter received both original and copied DNA then a band would first appear for a cell with both 14N and 15N. As the 14N continued to be incorporated into daughter cells a new band at only 14N would also form. This is a classic experiment first performed by Meselson and Stahl.

Basically analyse the sides of the bands between each generation, and see how it progresses while in the 14N media.

Question 10

What end can DNA polymerase add more nucleotides

Answer 10

3’ (3 prime)

Ie you can only extend DNA going from 5’ to 3’

Question 11

Which direction does the leading strand run

Answer 11

3’ to 5’

Question 12

What direction does the lagging strand run

Answer 12

5’ to 3’

Question 13

Why is it called the lagging strand

Answer 13

As it’s a slower process to synthesise due to the necessity of the Okazaki fragments

Question 14

What is the name of the enzyme that unwinds the dna strands during synthesis and its purpose

Answer 14

Topoisomerase – unwinds the DNA to make it accessible to other enzymes.

Question 15

Name the enzyme that splits the dna strands

Answer 15

Helicase – separates the two strands of DNA unwound by Topoisomerase.

Question 16

Name the enzyme that synthesises the new stand

Answer 16

DNA polymerase – adds nucleotides to the new DNA strand in a complementary fashion to the template strand.

Question 17

Same the enzyme that enzyme that positions the short rna primers on the lagging strand

Answer 17

RNA primase – adds short rna “primer” sequences (Okazaki fragments) to the lagging strand during DNA replication.

Question 18

Name the enzyme that joins the Okazaki fragments together

Answer 18

DNA ligase – joins Okazaki fragments together

Question 19

What is the central dogma of molecular biology

Answer 19

The central dogma of molecular biology describes the two-step process, transcription and translation, by which the information in genes flows into proteins: DNA → RNA → protein.

Question 20

What do restriction enzymes do

Answer 20

Cut both strands of DNA at/near specific recognition sites called restriction sites

Question 21

What’s gel elctrophoresis

Answer 21

A method for separation and analysis of macromolecules (DNA, RNA, Proteins) and their fragments, based on their size and CHARGE

Electric field separates negatively charged nucleic acids through a matrix of agarose, shorter molecules move faster and further through the pores of the gel.

Question 22

What are sticky ends

Answer 22

Caused when restriction enzymes cut DNA in an offset fashion, with an overhanging piece of single stranded dna.
Called sticky as can only form base pairs with dna that has the complimentary base pairs. (Comparable sticky ends)

Question 23

What enzyme repairs double stranded breaks (such as that caused by restriction enzymes)

Answer 23

DNA Ligase

Question 24

What is transformation

Answer 24

The process of adding foreign DNA into a cell

Question 25

What’s a cloning vector

Answer 25

A small piece of dna that acts as a backbone into which dna fragments can be inserted for cloning purposes.

Question 25

What are restriction enzymes

Answer 25

Enzymes that cut/cleave DNA at/near specific recognition sites within the molecule knows a restriction sites. They recognise a specific sequence of nucleotides and produce a double stranded cut in the dna

Question 26

What is recombinant dna

Answer 26

Dna made by lab methods (created artificially)

Question 27

What’re eukaryotic cells

Answer 27

Cells which have a membrane bound nucleus

Question 28

What’re prokaryotic cells

Answer 28

Cells that lack a membrane bound nucleus

Question 29

Tow methods for bacterial transformation

Answer 29

Chemical transformation:
Chill the cells in CaCl2 to permeabilise membrane
Heat shock prompts uptake of DNA

Electroporation:
Purify the cells to remove ions
Punch holes in the membrane with a high voltage shock

Remember after this to give cells time to recover before selection.

Question 30

4 main steps of dna cloning

Answer 30

1. Chosen piece of dna is cut from the source organism by restriction enzymes
2. Dna is pasted into a vector and the ends of the dna are joined with the vector dna by ligation
3. The vector is introduced in to a host cell by bacterial transformation and the host cells copy the dna along with their own dna, creating multiple copies of the inserted dna
4. The vector dna is isolated/separated from the host cell’s and purified.

Question 31

What is the purpose of PCR

Answer 31

Polymerase chain reaction amplifies a single copy of a segment of DNA (target dna) across several orders of magnitudes (thousands/millions of copies). Goal is to create enough copies so that it can be analysed in some other way.

Question 32

6 things needed in the solution for PCR

Answer 32

The target dna
Primers
Nucleotides
The enzyme DNA polymerase 
Buffer (to create optimum pH for enzyme)
MgCl2 for the enzyme to work

Question 33

3 steps in PCR

Answer 33

Denaturation- temperature is raised (>90degrees) to break the hydrogen bonds between the nucleotides (complimentary base pairs) and separate the two strands of DNA

Annealing- temp is lowered to 55-65degrees so that the primers bind to the target DNA sequences and initiate polymerisation. One primer binds to each strand so a forward and reverse primer is required

Extension- temp is raised slightly to 72 so that the DNA polymerase can synthesise the new strand, adding the free nucleotides to the template strand

The result of PCR is two double stranded sequences of target dna, each containing one newly made strand and one original strand
The 3 step temp cycle is repeated many times to get many copies of the target dna.

Question 34

What is the recommended length for a pcr primer

Answer 34

20 or more base pairs (bp)

Question 35

What is Gibson assembly

Answer 35

A molecular cloning method that allows for the joining of multiple dna fragments in a single isothermal reaction.

Question 36

Steps of Gibson Assembly

Answer 36

1. Create a 20-40 base pair overlap between the dna fragments to be joined by PCR
2. Add to Gibson assembly master mix and incubate at 50 degrees.
3. T5 exonuclease chews back the 5’ ends.
4. Overlap regions anneal
5. DNA polymerase then extend the 3’ ends, filling the gaps
6. DNA ligament then seals the remaining nicks.

Question 37

3 enzymes in the Gibson Assembly “master mix” buffer

Answer 37

T5 exonuclease
DNA Polymerase
DNA ligase

Question 38

What base pairs tie down the primer better

Answer 38

G-C are better than A-T, should be considered when designing where you want the primer to attach

Question 39

If you have an organism with a particular trait dependent on a single gene, how could you clone out and sequence the gene given you can’t sequence the whole organism, but know organisms with a similar trait

Answer 39

Find the sequences of the similar organisms online and find the gene responsible for the trait. Compare and come to a consensus of particular similarities that identify the gene, which can be used to prepare primers to sequence the organism around the area of the gene. PCR can then be used with more precisely designed primers to amplify out the region of genomic DNA you care about. Can then check the function of the gene by inserting it into a lab strain E. coli

Question 40

To order of magnitude, how many colonies do you expect on the selection plate

Answer 40

Each derived by one parent bacteria

Question 41

To order of magnitude, what is the efficiency of DNA uptake by cells in bacterial transformation

Answer 41

1 in 10^3 or 10^4

Question 42

Do cell populations grow much when under bacterial transformation

Answer 42

No, maybe a factor of W increase, not significant in order of magnitude calcs

This is due to the stress they have encountered in the ice- heat shock cycle

Question 43

What is Crispr/cas9

Answer 43

A technology that amenable geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence. Currently the simplest, most versatile and precise method of genetic manipulation.

Question 44

What are the two key molecules in crispr

Answer 44

Enzyme Cas9 and a piece of guide RNA

Question 45

What were the old methods of altering genes before crispr

Answer 45

Chemicals or radiation

They had no way to control where the gene mutation occurred