Overview/intro Flashcards
What are the bases in DNA
A,C,G,T
What are the bases in RNA
A,C,G,U
What molecules are involved in storage, transfer and processing in the central dogma of biology
Storage: DNA
Transfer: RNA
Processing: Protein
What makes DNA good at information storage
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.
What makes RNA good at its job of transfer
short lived so suitable for transient expression.
What makes proteins good at their job of processing
complex three-dimensional structure allows for the performance of a wide variety of chemical and structural tasks.
Why functions would RNA need to be the sole precursor to life and why would it have been appropriate?
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.
What are the 3 different configurations in which DNA could conceivably replicate? And which was it?
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
How could you confirm experimentally what mode of replication happens in DNA
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.
What end can DNA polymerase add more nucleotides
3’ (3 prime)
Ie you can only extend DNA going from 5’ to 3’
Which direction does the leading strand run
3’ to 5’
What direction does the lagging strand run
5’ to 3’
Why is it called the lagging strand
As it’s a slower process to synthesise due to the necessity of the Okazaki fragments
What is the name of the enzyme that unwinds the dna strands during synthesis and its purpose
Topoisomerase – unwinds the DNA to make it accessible to other enzymes.
Name the enzyme that splits the dna strands
Helicase – separates the two strands of DNA unwound by Topoisomerase.
Name the enzyme that synthesises the new stand
DNA polymerase – adds nucleotides to the new DNA strand in a complementary fashion to the template strand.
Same the enzyme that enzyme that positions the short rna primers on the lagging strand
RNA primase – adds short rna “primer” sequences (Okazaki fragments) to the lagging strand during DNA replication.
Name the enzyme that joins the Okazaki fragments together
DNA ligase – joins Okazaki fragments together
What is the central dogma of molecular biology
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.
What do restriction enzymes do
Cut both strands of DNA at/near specific recognition sites called restriction sites
What’s gel elctrophoresis
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.
What are sticky ends
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)
What enzyme repairs double stranded breaks (such as that caused by restriction enzymes)
DNA Ligase
What is transformation
The process of adding foreign DNA into a cell
What’s a cloning vector
A small piece of dna that acts as a backbone into which dna fragments can be inserted for cloning purposes.
What are restriction enzymes
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
What is recombinant dna
Dna made by lab methods (created artificially)
What’re eukaryotic cells
Cells which have a membrane bound nucleus
What’re prokaryotic cells
Cells that lack a membrane bound nucleus
Tow methods for bacterial transformation
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.
4 main steps of dna cloning
- Chosen piece of dna is cut from the source organism by restriction enzymes
- Dna is pasted into a vector and the ends of the dna are joined with the vector dna by ligation
- 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
- The vector dna is isolated/separated from the host cell’s and purified.
What is the purpose of PCR
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.
6 things needed in the solution for PCR
The target dna Primers Nucleotides The enzyme DNA polymerase Buffer (to create optimum pH for enzyme) MgCl2 for the enzyme to work
3 steps in PCR
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.
What is the recommended length for a pcr primer
20 or more base pairs (bp)
What is Gibson assembly
A molecular cloning method that allows for the joining of multiple dna fragments in a single isothermal reaction.
Steps of Gibson Assembly
- Create a 20-40 base pair overlap between the dna fragments to be joined by PCR
- Add to Gibson assembly master mix and incubate at 50 degrees.
- T5 exonuclease chews back the 5’ ends.
- Overlap regions anneal
- DNA polymerase then extend the 3’ ends, filling the gaps
- DNA ligament then seals the remaining nicks.
3 enzymes in the Gibson Assembly “master mix” buffer
T5 exonuclease
DNA Polymerase
DNA ligase
What base pairs tie down the primer better
G-C are better than A-T, should be considered when designing where you want the primer to attach
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
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
To order of magnitude, how many colonies do you expect on the selection plate
Each derived by one parent bacteria
To order of magnitude, what is the efficiency of DNA uptake by cells in bacterial transformation
1 in 10^3 or 10^4
Do cell populations grow much when under bacterial transformation
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
What is Crispr/cas9
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.
What are the two key molecules in crispr
Enzyme Cas9 and a piece of guide RNA
What were the old methods of altering genes before crispr
Chemicals or radiation
They had no way to control where the gene mutation occurred
