Biochem Practicals

How do some bacteria manage not to digest their own DNA but still digest transformed DNA

• Modify by methylation of A and/or C

• One strand is immediately methylated after replication so it can be protected whilst other strand is methylated

Why use two different restriction enzymes when cloning into a DNA fragment into a plasmid

• Directional

Why do we want directional cloning?

• Expression of correct protein

• Ensure the correct strand is transcribed in in vitro transcription

How to check that cloning has been directional without sequencing the clones?

• Use restriction enzymes that cut asymmetrically within the plasmid

• Know exactly where they cut in the insert and in the plasmid

• Draw a map

• Separate fragments on agarose

• Compare the expected size of fragments for each map to the size of fragments displayed on the agarose

Reasons why PCR has failed, besides just forgetting to add any components

• DNA template degraded / contaminated / too little

• Primers form secondary structures or dimers

• Elongation cycling time is too short

  • To little Mg2+ so too much repulsion

Why might you obtain a series of PCR products when only one is expected? How is this avoided?

• Non-specific priming

• Increase annealing temperature

• Decrease Mg2+ concentration to increase repulsion

  • Change primer

Why is RT often used before qPCR

• Forms cDNA so that it can be amplified by PCR

• Amount of DNA produced after a set number of cycles shows how much DNA was started with

What are two common methods for detection of products in qPCR

• Fluorescent dye that non specifically intercalates into dsDNA (SYBR green)

• Sequence specific DNA probes with a 5’ fluorophore and 3’ quencher (fluorophore removed by 5’ → 3’ exonuclease activity of DNA pol)

What control is required for SYBR green?

• Binds to any dsDNA in the sample

  • Verify that only the product of interest is amplified

Where is RT derived from?

• AMV

  • MMLV

How to use RT to form DNA from RNA

• Anneal with oligo(dT) primer to bind to the poly(A) tail

• Reverse transcriptase will continue

ignore

• okayyay

What is a housekeeping gene used for in qPCR

• Determined as a gene that does not change expression levels when given the same treatment that is altering the expression of the protein in question

• Reports the relative amount of starting cDNA for that sample

• Allows comparison in change of expression levels as change in fluorescence could just be different amounts of template started with

Rationalise the effects of NaCl and MgCl2 on the stability of dsDNA

• Reduce repulsion between phosphates in the backbone

• Mg2+ more effective as higher charge density

Rationalise the effects of urea on the stability of dsDNA

• Urea competes for the hydrogen bonds between the base pairs

  • Reduces attraction between bases

How does separation achieved by gel electrophoresis differ from gel filtration

• Gel filtration: larger molecules move faster as they pass rapidly around matrix beads and do not have to squeeze through them

  • Gel filtration: smaller molecules move slower as they are able to move space both within and outside the porous matrix

Purpose of a sodium phosphate wash buffer in gel filtration

• Removes unbound proteins

Purpose of a sodium phosphate + imidazole wash in gel filtration

• Competes for Ni2+ sites, knocking off any contaminating proteins that have unpaired electrons used in nickel binding or external his residues

Techniques for measuring specific interactions between DNA and DNA-binding proteins

• DNA footprintng

• Modification interference e.g. methylation protection

• ChIP

• UV-crosslinking

Explain blue-white selection

• Beta-galactosidase breaks down X-gal into a blue substrate

• MCS of pBluescript is inside LacZ’ gene

• Inserting gene into LacZ’ inactivates gene: no functional beta-galactosidase

• Add IPTG and X-gal

• Plasmids with insert will be white, without insert will be blue

Explain how Bradford assays work

• Coomassie dye has a red and a blue form

• Dye binds to proteins, which stabilizes the blue form

• Amount of complex present in the solution can be estimated using absorbance reading at 595nm

• Amount of complex also proportional to amount of protein present in sample

• Make a standard curve using known amount of BSA

• Intraplate absorbance reading of sample on standard curve

What is a cDNA library?

• cDNA prepared using RT on mRNA

• Collection of cloned cDNA fragments inserted into E.coli host cells

• Can screen library for clone of interest by plating onto agar plates and then transferring colonies that grow onto a membrane probed with specific radiolabeled DNA probes to the sequence of interest

• Where radioactivity is can be extrapolated back to the colony position on the original agar plate

Simply, what is ChIP used for?

• Determine whether specific proteins are associated with specific genomic regions

  • E.g. transcription factors associated on promoters or other DNA binding sites

Describe the prosses of NChIP (native chip)

• Chromatin in the nucleus under native conditions

• Formaldehyde cross linking reversibly cross-links protein to chromatin in the cells

• DNA sheared by micrococcal nuclease digestion into fragments 300bp, still associated to protein

• Antibodies to protein of interest coupled to protein A beads immunoprecipitated the protein of interest, which is still cross-linked to the DNA fragment

• Cross-links are reversed

  • DNA purified and analyzed with PCR and direct high-throughput sequencing

Simply, what does DNA footprinting do?

• Determines where protein binding protects DNA from attack by nucleases or chemical probes

• Determines precise binding site of a protein on a fragment of DNA

Describe how DNA footprinting is carried out

• DNA is one end-labelled using 32P

• Protein incubated with dsDNA

• Mildly digested with DNAase, each DNA molecule is cut once

• Separate on agarose

• Visualise radioactivity

• DNA without protein will have a continuous fragment ladder (cuts at a random points on every strand)

  • DNA and protein will have a gap (footprint) where DNA has been protected and remains associated with protein

What does FISH stand for?

• Fluorescence in situ hybridization

What does FISH do?

• Detect and localise presence of specific DNA sequences on chromosomes

  • Detect and localise specific RNA targets inside cells

• Gives the cool different coloured fluorescence diagrams

Describe how FISH is carried out

• Cells are fixed and permeabilized

• Oligonucleotides complementary to sequence of interest are fluorescently labelled

• Incubated

• Probes bind to mRNA

• Fluorescent microscopy visualizes where labels are in cells

  • Can use different probes with different fluorescent labels to create a colourful diagram

How is FISH used clinically?

• Detect genetic conditions by testing if the gene sequence is present in chromosome

• Evaluate progression or remission of a disease e.g. cancer

What is the basis of gel filtration of a protein mixture?

• Small molecules can penetrate the pore system of beads in the matrix, causing retention that slows their path

  • Large proteins can’t penetrate pore system so only enter interbead space and elute first

Agarose gels

• Separate mixed masses of DNA or RNA

• Small molecules move fastest

• Can then be transferred to a suitable membrane for southern/northern blots

Formaldehyde agarose

• Denaturing agent in RNA separation gels

SDS-PAGE gels

• Separate proteins according to size

  • Denaturing

Native PAGE

• Separation of proteins by size and shape as non-denaturing

• Used in EMSAs to analyze nucleic acid: protein complexes

8M urea gels

• Separate DNA or RNA by molecular weight with higher resolution

What does iCLIP stand for?

Individual-nucleotide resolution cross linking immunoprecipitation

Simply, what does iCLIP do?

• Identify protein-RNA interactions

Describe the process of iCLIP

• UV light covalently cross-links RNA with any of its interacting protein molecules

• Cell is lysed

• Immunoprecipitate protein-RNA interactions (antibody to protein with protein A beads)

• RNAs labelled using 32P

• SDS-PAGE

• Transferred to nitrocellulose membrane for Western blotting

• Radioactivity detected

• Radiolabelled complexes removed from membrane, treated with protease to extract RNA

• RT to get cDNA

• High throughput sequencing to pinpoint crosslink sites

What does RIP stand for?

• RNA immunoprecipitation

What does RIP do?

• Antibody-based technique to map in-vivo RNA-protein interactions

• Fix antibody against protein of interest against side of well

• Mix protein with various RNA, add to well

• Repeat washings so only protein of interest of left

• RT to extract cDNA from RNA bound in protein

• PCR or seq

How does a pull down assay work?

• Tag protein of interest with his tag or GST tag

• Immobilize protein of interest on a column of nickel (for his tag) or glutathione sepharose (for GST tag)

• Add protein mixture

• Wash thoroughly

• Only protein complex interacting with POI remains bound

• Elute using imidazole or glutathionine

  • Analyse SDS-PAGE or mass spec

Briefly, how does RNAi knockdown gene expression?

• siRNA forms a complex with Ago2 RE that is inside RISC

• Ago2 catalyzes endonucleolytic cleavage of target RNAs when siRNAs bind

How would you downregulate expression of protein X using RNAi?

• Make 21bp siRNA that is specific for mRNA of protein X introduced to cell as dsRNA (guide stand and passenger strand with 2bp overhangs)

• Guide strand interacts with Ago2 within RISC

• Guide strand binds specifically to protein X mRNA

• Ago2 cleaves mRNA that is bound to the siRNA

  • Rest of the mRNA undergoes further decay using cellular pathways

How would you check that you have knocked down a protein expression when you’ve used RNAi?

• Western blot with radioactive probes for protein X

  • Use sample treated with specific siRNA and a control sample that has used a random siRNA

Why is using RT-qPCR not very reliable as a method of measuring if RNAi has knocked down mRNA expression

• Does not report on actual protein level

• Depends on half life of RNA as well

Briefly, how is Southern blotting carried out?

• Separate DNA on agarose

• Transfer to a membrane

• DNA of interest is detected using hybridization of a specific detectable probe

• Visualised using appropriate method

Assumptions of the Michealis Menten equation?

• SCANC: spontaneous, constant, allostery, negligible, cooperativity

• No spontaneous creation of product

• [substrate] »» [enzyme] so that substrate concentration can be assumed constant throughout the assay

• Neither substrate nor product is allosteric modulator of the enzyme involved

• [product] negligible so rate of reverse reaction is 0 when measuring

• No co-operativity

What makes fluorogenic compounds more sensitive than chromogenic ones?

• Fluorescence is always a different wavelength to the absorbed wavelength

• Allows detector to be set to a different wavelength to the illumination of the sample

• Don’t have to remove background intensity

• Less “noise”

How to check for possible non-enzymatic hydrolysis of the substrate

• Control with no enzyme

• Measure background

Convert from mg/mL to g/L

• They are the same

Convert from g/L to M

g/L / Mr

State 1 mitochondria

• Mitochondria alone

• Slow as ETC inhibited by high pmf

State 2 mitochondria

• Substrate added

• Slight increase in rate as ETC running

• No ADP so ATPSase not running

• Pmf not dissipated

State 3 mitochondria

• ADP added

• Pmf can be dissipated via ATPSase

• Inhibition released

State 4 mitochondria

• Ran out of ADP

• Pmf accumulates again

• Rate slows, roughly same rate as in state 1

What is done experimentally after state 4 and why?

• Add uncoupler e.g. FCCP

• Rapidly dissiptates pmf: is a proton transporter along the electrochemical gradient

• Rate increases rapidly

• To reach zero oxygen on a graph being generated so that mmol o per square can be calculated

How to calculate mmol O per square on the mitochondria graphs

• Number of squares between start and when zero oxygen is reached after uncoupler is added is volume of electrode chamber x air saturated concentration x2 (O not O2)

Inhibitors for each of the complexes, ATPSase and uncouplers

• RACOF

• I : rotanone

• III: antimycin

• IV: cyanide

• ATPSase: oligomycin

• Uncoupler: FFCP

What are the substrates that feed electrons into complex I and II?

• I: NADH (hard to use experimentally, malate/glutamate used instead)

• II: Succinate

Describe the movement of electrons through the etc

• Substrate feeds into CI OR CII

• Feeds into CIII

• CIV forms water from oxygen

How many protons do each of the complexes pump into the intermembrane space

• I: 4

• II: None

• III: 4

• IV: 2

How to calculate experimental P:O

mol ADP consumed in state 3 / mol O consumed in state 3

How to calculate theoretical P:O

H+:O/H+:P or 10 when using CI, 6 using C2, all over 8/3 + 1

How to analyse P:O ratios

• 2-3, doesn’t look like there’s anything wrong with the complex being used

How to calculate RCR, and what is it used for?

• rate of state 3 / rate of state 4

• To see if membrane is damaged (S4 would run too fast as damaged membranes are already partially uncoupled, so RCR would be too low)

Range for RCR analysis

4-8 Good, membranes coupled

<4 bad, damaged membranes

Why use malate / glutamate instead of NADH to feed into complex I?

• NADH hard to use experimentally, not very stable

• Easier to buy, more stable

Why are mt rested and fasted before experiments?

• Deplete electrons

• Whatever is added experimentally is the only source of electrons

Analysis if experimental P:O > theoretical

• Air bubble in chamber

Analysis if experimental P:O < theoretical by about 10-15%

• Experimental error (pipetting)

Analysis if experimental P:O < theoretical by more than 15%

• Damaged complex or mutation

Lineweaver-Burk equation

1/V = (Km/Vmax) * (1/[S]) + (1/Vmax)

What should be on the axes in Lineweaver-Burk plots?

• x: 1/[S]

• y: 1/V

Define Vmax

• Maximum ROR when enzyme is fully saturated with substrate

Define Km

• Substrate concentration at which reaction rate is half of Vmax

Define Kcat

• Number oof substrate molecules converted to product by single enzyme molecule per unit time when enzyme is in fully saturated conditions

Equation for kcat

Kcat = Vmax / total enzyme concentration

Draw a diagram of where the forward and reverse primers bind

https://ibb.co/wNsCX7Hv

Start codon (DNA)

• ATG

Stop codons (DNA)

• TGA, TAA, TAG

Reccomended G/C content of a primer

• 40-60%