Dna Analysis

Question 1

Define electrophoresis

Answer 1

• Movement in an electric field

Question 2

Why is electrophoresis used in mb

Answer 2

• Used to analyse by separating molecules
• Used to purify
• Size markers can be used to estimate molecular weight

Question 3

Types of molecules commonly analysed by electrophoresis

Answer 3

• Protein (+ or – charge)
• RNA -ve
• DNA -ve

Question 4

Forces that affect electrophoresis

Answer 4

• Velocity affected by electric field and electrophoretic mobility
• Electrophoretic mobility affected by solutes charge, solutes radius and environments viscosity
Increases with charge
Decreases with radius and viscosity

Question 5

How do forces affect analytic techniques in electrophoresis

Answer 5

• Can increase size of electric field to speed up electrophoresis
• Done by changing the voltage
• Electric field calculated by (V/d)
• If you try to speed it up it may heat up the media, causing it to melt
• The heating can also cause less discrete lines
• Can determine size of fragments using size markers of known molecular weights

Question 6

How can understanding forces affecting electrophoretic mobility be exploited in analysis

Answer 6

• Plasmids have different conformations
• Migration depends on shape (radius)
• Tightly supercooled plasmids will move the fastest

Question 7

Denaturing SDS polyacrylamide gel electrophoresis (SDS page)

Answer 7

• Used to minimise the effect of shape and charge differences
• The main factor that influences migration is size
• Cross linked gel of polyacrylamide used as the matrix for proteins to pass through
• Gel prepared by polymer is action of monomers so pore size of gel can be adjusted so t is small enough to retard (delay or hold back) the protein of interest
• Proteins dissolved in solution containing highly -Ve charge detergent SDS
• Detergent binds to hydrophobic regions of protein, causing it to unfold
• Individual protein molecules released from associations with other proteins or lipids
• Reducing agent (beta-mercaptoethanol) added to break any S-S bonds, frees all polypeptides in multi subunit proteins
• -ve charged detergent molecules mask proteins intrinsic charge
• Proteins of same size tend to move at similar speeds as they are unfolded so shapes are the same and they bind same amount of SDS so have same amount of -ve charge
• Large proteins have larger electrical force but also larger drag
• These two factors don’t cancel out as the polyacrylamide gel acts as a molecular sieve that retards large proteins much more than small ones
• This separates the proteins into discrete bands

Question 8

Non-denaturing polyacrylamide gel electrophoresis (native PAGE)

Answer 8

• Used to study molecular interactions
• Usually at low voltage to prevent denaturing
• Gels without denaturant
• Bands with different mobilities can be observed, corresponding to each conformational state
• ^ if exchange rate (from bound to free) is slow relative to rate of transport

Question 9

Agarose properties

Answer 9

• Agar is extracted from red algae
• Gelifying agent
• Agarose and agaropectin make up agar
• Agarose better for electrophoresis
• Made up of galactose and anhydroglaactose
• Reversible gel-solution
• Liquid until 40/52 degrees C
• Put powder in water/buffer and heat to near boiling point
• Mostly uncharged
• Low binding to proteins, nucleic acids and stains
• Moderate resistance to hydrolysis
• Solid at very low concentration (>0.15%) – allows larger fragments to be separated
• Relatively inexpensive
• Non-toxic

Question 10

How is agarose used as electrophoresis medium

Answer 10

• Usually submarine gels – horizontal and submerged in buffer
• Chromosomes can be separated with contour clamped homogenous electric field (CHEF) gel electrophoresis
• Need highly purified agarose
• E field switched from + to -, rotated around gel causing molecules to zig-zag across gel

Question 11

What is polyacrylamide

Answer 11

• Synthetic gel
• Synthesised from monomers
• Main monomer is acrylamide
• Tetramethylethylethylenediamine (TEMED) and ammonium persulphate (APS) react to make radicals that cause monomers to polymerise
• Methylenebisacrylamide created bridges between linear polymers

Question 12

Why is polyacrylamide gel used for electrophoresis

Answer 12

• By changing proportions you can have a tighter network i.e. more bridges makes it tighter
• Acrylamide for smaller fragments – agarose for larger
• Simple reactions controllable at room temp
• Reproducible gel characteristics by using known conc of monomer
• %T= total conc of acrylamide and bisacrylamide , %C= conc of bisacrylamide
• Uncharged
• Low binding to proteins, nucleic acids and stains
• Resistance to hydrolysis
• High strength gels allows thin configurations
• Relatively inexpensive
• Toxic monomers but non-toxic polymers
• Polyacrylamide has better focusing and resolution than agarose

Question 13

How is polyacrylamide used in electrophoresis

Answer 13

• Vertical slab
• Acrylamide cast between 2 glass plates
• Not submarine
• Mostly used for electrophoresis of proteins or SHORT polynucleotides
• Used in SDS – page
• Lower % ‘stacking gel’ that is also low pH
• Discontinuous ‘separating’ gel with higher pH
• Proteins squish together in stacking gel, focused
• Gives thin, sharp bands, higher resolution

Question 14

How to use electrophoresis to estimate relative size of nucleic acids and proteins

Answer 14

• Modulating by altering gel concentration (size of molecular pores) enables you to adapt the analysis for molecules of different size range
• Can create calibration curve to estimate size of molecules
Plot log of size in BP against distance of migration
Ferguson plot - log mobility = log electrophoretic mobility against retardation coefficient x [gel]
• For DNA, denatured RNA, protein-SDS complexes
• Charge density (q) is constant
• Electrophoresis (especially in agarose) not very precise so be careful with number of SF

Question 15

Most common ways of detecting nucleic acids and proteins in gel media

Answer 15

• DNA and RNA- ethidium bromide, Gel Red, Sybr, Silver stain
• Proteins- Coomassie Blue, Sybr, silver stain

Question 16

How do nucleic acid and protein stains work

Answer 16

• Ethidium bromide absorbs UV and emits orange/ red light, concentrates in the DNA
• Sybr absorbs blue light and emits green light
• Coomassie blue used to stain gel after electrophoresis

Question 17

Pros and cons of nucleic acid and protein stains (ethidium bromide, gel red, sybrsafe)

Answer 17

• Ethidium bromide risk factors:
• DNA intercalating dye-mutagen
• Excitation max is UV, UV is a mutagen and can cause burns
• Gel red:
• Analogue of ethidium bromide with lower cell penetration ability – reported less toxic than ethidium bromide
• SybrSAFE:
• DNA intercalating gel- reportedly less mutagenic than ethidium bromide
• Excitation max- blue light- harmless

Question 18

What is hybridisation of nucleic acids (NAs)

Answer 18

• Hybridisation = renaturation = annealing
• 2 complementary single stranded dna or rna molecules bond together to form a double stranded molecule
• Reverse is: denaturation/ dissociation/melting

Question 19

Factors influencing hybridisation in solution

Answer 19

• Length of dna
• Composition of dna
• Greater proportion CG= higher temp needed to melt
• % of complementary bases
• Sometimes strands form when not all bases are complementary – lower temp to melt
• Composition of medium (pH,ions,denaturing agents)
• Temperature

Question 20

DNA denaturation

Answer 20

• Absorption curve can be used to show the loss of hybridisation in DNA, called a melting curve
• Tm= melting temperature, when 50% melted and 50% hybridised
Dentured dna has higher relative absorbance in sepectrophotometer

Question 21

Which molecular biology techniques rely on hybridisation

Answer 21

• Filter or membrane hybridisation- a technique that prevents parental strands re-annealing
• E.g. southern, northern, colony hybridisation, western
• Microarrays- hybridisation analysis of thousands of dnas (genes) simultaneously
• In situ hybridisation- allows localisation of target dna/rna in tissue e.g. fluorescence in situ hybridisation (FISH), chromosome painting)
• PCR
• DNA sequencing

Question 22

Southern hybridisation (type of membrane hybridisation)

Answer 22

• Capillary transfer of dna or rna
• Electrophoresis through gel, membrane (nitrocellulose or nylon) on gel, lots of paper on top of membrane, buffer above paper moves through paper and transfers DNA from gel to membrane
• Used to analyse DNA (northern is rna)
• DNA is separated by size on an agarose gel and transferred onto nitrocellulose, nylon or PVDF membrane
• Radioactive labelled dna fragment (probe) is incubated with the membrane and the hybridised bands are detected by exposing an X ray film to the radioactive ‘bands’
• Can be used to detect specific dna sequence in dna samples
• Probe anneals to complementary sequences
• Autoradiography reveals places where probe has hybridised
• In southern blot the gel is soaked in NaOH or alkaline solution to denature dsDNA to ssDNA before membrane transfer
• Blotting: transfer of nucleic acids to a solid membrane support

Question 23

Western blotting

Answer 23

• Electrophoresis moves proteins upwards towards positive charge so they are pulled up out of gel to transfer onto membrane (field applied up and down instead of side to side like in southern)
• Nitrocellulose paper or nylon membrane
• Electrophoretic transfer of proteins
• Membrane then soaked in solution of labelled antibody to reveal protein of interest
• Used for protein quantification, protein structure, protein modifications
• Southern-dna target- ssDNA or RNA probe
• Radioactive label and mix with membranes with target on
• Northern-RNA target- ssDNA or RNA probe
• Western-protein target- antibody probe

Question 24

Colony hybridisation

Answer 24

• If a bacteria colony or plaque is potentially carrying a fragment of interest, it can be screened by hybridisation
• Put filter on top of colonies, denature colonies and hybridise with probe, colonies detected by probe

Question 25

Microarrays

Answer 25

• Hybridisation analysis of 100s of thousands of targets simultaneously
• First developed to analyse global gene expression
• Probe not labelled
• DNA probe complementary to genes (cDNA) of interest is generated and positioned in microscopic quantities on solid surfaces at defined positions
• Target dna generated from the sample rna is fluorescently tagged and allowed to hybridise to ‘dna chip’
• Two populations of target dna (labelled with different fluorophores) can be analysed simultaneously
• Detected by monitoring fluorescence following laser excitation
• Different types of sequences of nucleic acids on solid surbstrate
• Usually put on glass slide that is attractive to dna
• Can do at high densities

Question 26

In situ hybridisation

Answer 26

• Samples cut into sections
• RNA or DNA is fixed and probed in situ i.e. in the position it occupied in the living tissue
• Hybridised with radioactive ss NA probe, followed by autoradiography
• FISH (fluorescence in situ hybridisation) proteins are detecte with immunofluorescence, using antibody probes, examine with fluorescence microscope to see location and intensity

Question 27

Algorithms to estimate Tm of NA sequences

Answer 27

• Tm increases with:
• Increasing c-g content
• Increasing na+ conc
• Decreasing formamide conc (a denaturing agent)
• Increasing length of hybrid
• For short dna (oligonucleotides) we can used algorithms to estimate melting point
• Tm(0C) = 4x (no of G andC)+2x(no.A and T)
• Tm decreased by approximately 10C for every 1% mismatch

Question 28

Hybridisation terminology

Answer 28

• Formation of a double stranded helix or hybrid usually between a probe and its complementary target
• Stability of hybrids – DNA/DNA<DNA/RNA<RNA/RNA
• Double stranded DNA least stable
• It is possible for NA strands that are not fully complementary to hybridise
• Stringency- % homology between the stands
• Low stringency- temp of hybridisation much lower than Tm for perfect matc
• High- temp of hybridisation closer to Tm for a perfect match
• Homologous hybridisation- probe + target contain identical sequences – high stringency
• Heterologous hybridisation- probe and target contain similar but non-identical sequences
• E.g. finding genes in humans using drosophila probe- lower stringency

Question 29

Use of term homology in MB

Answer 29

• Homologous sequences share a common evolutionary origin
• Seeing they’re similar doesn’t tell you if they’re homologous or not
• Nucleotide sequence can be described as having a certain % sequence identity
• Cannot describe sequences in terms of % homology
• They either are homologous or they aren’t

Question 30

Basis of Sanger sequencing

Answer 30

o Uses dna polymerases
o Sequencing by synthesis
o Primer binds to end of sequence- need to know what this sequence is
o Radioactive primer
o Add buffers and nucleotides
o 4 separate reactions adding a dideoxy terminator for each base at a low concentration
o Polymerase will sometimes incorporate this molecule, then sequencing will stop
o Each reaction has a different ddNTP (ddATP, ddGTp etc.)
o So in tube with ddATP each fragment has marked locations of T nucleotides
o Analyse by electrophoresis
o Used to use radioactive labels, now use fluorescent
o In fluorescent, all ddNTP added at same time, each with different label
o The dideoxyterminators have no oh group on the sugar so terminate polymerisation

Question 31

How has Sanger sequencing evolved over time

Answer 31

o Now use cycle sequencing which is still Sanger
o Not exponential amplification like PCR
o Similar to PCR temps, uses Taq polymerase
o Denature dna, anneal primer, extend and terminate
o At end of cycle denature and start again
o Get a lot more product
o No priming from both ends, only one end
o Primer has dye and terminators also have dye
o Sanger used to have to be read off electrophoresis gel
o Now uses virtual electropherogram

Question 32

How to interpret electropherograms

Answer 32

o 4 colours
o Peaks correspond to different bases
o Height of a peak is directly proportional to fluorescence emitted by the fragment
o Different coloured peaks for different bases
o DdNTP terminating chain will emit signal that is detected

Question 33

Sequencing by synthesis protocol used by illumina/solexa

Answer 33

o Size of a microscope slide
o Dna loaded into wells
o Shear dna to end up with short double stranded dna
o Ligation with adaptors that end up on ends of molecule
o Fragments unknown with known adaptors
o Bind single stranded fragments randomly to the inside surface of the flow cell channels
o Target dna denatured and hybridises to dna on glass
o Add unlabelled nucleotides and enzyme to initiate solid-phase bridge amplification
o Dna folds over and binds to other complementary primer

o The enzyme incorporates nucleotides to build double-stranded bridges on solid-phase substrate
o Denaturation leaves single stranded templates anchored to the substrate
o Copies of dna end up in clusters on glass
o First sequencing cycle begins by adding four labelled reversible terminators, primers and dna polymerase
o Polymerise complementary strand using fluorescent nucleotides that are reversible terminators
o Scan arrays, machine sees different colours corresponding to where the base has been added
o For the next cycle, terminator has to be unblocked

Question 34

3rd gen high throughput dna sequencing by pacBio

Answer 34

o Zero mode waveguide
o Sequencing in minuscule well
o Bound dna polymerase at bottom of well
o As dna is synthesised, behaviour of light that is shone from underneath changes
o One molecule at a time
o Read as sequencing occurs

Question 35

3rd gen sequencing by oxford nano pore

Answer 35

o Very cheap to sequence molecules e.g. whole plasmid
o Arrays on membranes
o Proteins are added that are specifically engineered to make nano pores
o Complex proteins that also contain proteins that can read dna
o Double stranded dna so one end can be threaded through the pore driven by electrophoresis
o We can detect currents in the arrays
o Behaviour of current depends what base is at a specific point in the threading through

Question 36

Impacts of developments in dna sequencing on science and society

Answer 36

o Cost of sequencing genomes decreased dramatically when 2nd gen sequencing (illumina) was developed
o In 2000 only a few genomes had been sequenced, mostly bacteria
o New discipline developed - metagenomics – sequencing genomes of a community of organisms inhabiting a common environment
o Now ancient dna is sequenced
o Now way more genomes are sequenced
o Now the challenge isn’t sequencing, its bioinformatics

Question 37

Essential process of dna polymerisation

Answer 37

• Start with nucleic acid template
• DNA polymerase recognises portion of dna strand that needs to be double stranded
• DNA polymerase catalyses formation of phosphodiester bonds between 3’ end and nucleotides
• Works from 5’ to 3’
• Primer primes synthesis of complementary strand
• Primer is short sequence of dna, also called an oligonucleotide

Question 38

Brief pcr cycle

Answer 38

• Starts with target DNA sequence as a template
• Denature dna by warming solution
• Short primers complementary to part of target sequence anneal to target
• Usually in high abundance so equilibrium driven towards binding target
• Temp is lowered to allow hybridisation to occur
• DNA polymerase in solution with nucleotides and buffer controlling pH
• DNA polymerase makes complementary strands
• Oligonucleotide incorporated into product
• Cycle repeats
• Heat to denature again
• Get another set of double stranded molecules produced

Question 39

Pcr cycle with temperatures

Answer 39

• Start on ice at 4 degrees
• Don’t need to denature for more than 1-2 minutes
• Denatured at 92-95 degrees C
• Put in thermal cycles
• 55 degrees for annealing, temp can change depending on oligonucleotides
• Annealing very brief
• 72 degrees optimal temp for polymerisation of Taq polymerase
• Time depends on length of dna and type of polymerase
• Extend time if you want a full length of dna

Question 40

Importance of thermostable polymerase enzymes

Answer 40

• Exploit dna polymerases from thermophilic bacteria
• Not denatured by heating of dna strands
• I.e. don’t have to add more polymerase after every heating cycle

Question 41

How to quantify DNA target template

Answer 41

Y=A x 2^x
Y = final amount dna
A = initial amount dna in sample
X =number of pcr cycles
• Measure intensity of fluorescent signal from SYBR green which intercalates into dsDNA
• CT (cycle threshold) is number of cycles needed for signal to be detected above background
• The fold difference in amount of dna in 2 samples is 2^-deltaCT
• Exponential phase starts at 1 you just have so little material and product you wont detect it
• Exponential phase finishes as things run out
• Depends on concentrations of nucleotides and primers
• Also crowding of product
• Eventually enzymes wil degrade due to cycling
• Conditions become sub optimal
• Can use graph to quantify dna
• Put threshold as soon as you can detect product in exponential phase
• Because of saturation point, 2 lines should come to same level

Question 42

Compare types of thermostable dna polymerases

Answer 42

• Taq polymerase is fast but makes lots of mistakes, high error rate
• Some enzymes have proof reading, they will check if they are making a correct complementary strand
• Some are mixtures of different polymerases
• Error rate is important in cloning as you need an exact sequence, a mutation would be disruptive

Question 43

List pcr applications

Answer 43

• Detection/amplification of minute amounts of target dna – important in forensic science
• Quantification of nucleic acids
• Modification of target DNA (mutagenesis)
• Can introduce targeted mutations as oligonucleotides don’t need to be completely complementary to target
• Molecular cloning
• Sequencing
• Genotyping
• Paternity testing
• Detection of infectious disease

Question 44

How can pcr be used to introduce targeted mutations in dna

Answer 44

• Site-directed mutagenesis
• Uses primers that contain the desired mutation
• DNA in plasmid form
• PCR by using 2 primers that go in opposite directions
• One primer has a mutation
• Linear dna is made circular with a dna ligase
• Use restriction enzyme to get rid of template dna
• Use DpnI, acts on methylated DNA
• Plasmids from bacterial DNA will be methylated
• Sequence made by PCR wont be methylated
• Only template dna is degraded

Question 45

What is meant by reverse complementarità in DNA and rna

Answer 45

DNA strands are anti parallel
DNA complementarity is determined by Watson and crick base pairing of complementary bases
The reverse complement of a dna sequence is formed by reversing the letters, interchanging A and t and interchanging C and g

Question 46

Biological function of restriction endonucleases in nature

Answer 46

Bacteriophages:
Single phage infects a single bacterium
Phage reproduces to give 50-100 progeny
Infected cell lyses and released phages can infect surrounding cells
Phages reproduce in surrounding cells
Infected cells lyse and released phages can infect surrounding cells to produce a cleared area or plaque
Can add dilute suspension of phages to bacterial colony, spread on plate and incubate overnight,
If phage growing on strain B is added to K, K is less susceptible
The phages from the plaque in K escaped restriction i.e. the phage is not degraded
If you isolate these phages and add to K and B, now B is restricting and K is more susceptible i.e. the phage has learnt to avoid restriction
Restriction enzymes give bacterial immunity
Restriction system encodes an endonuclease and a dna methylase

Question 47

How is dna hydrolysed

Answer 47

Hydrolysed by nucleases- enzymes that break phosphodiester bonds
Many show specificity for the sugar backbone so there are Dnases, Rnases and non-specific nucleases
May be specific for single strands or double strands or be non specific
Restriction sites usually 4-8 bp long
Palindromic restriction sites
Differ by where in the phosphodiester bond they cut, leaving either 5’ phosphate and 3’ hydroxyl or 5’ hydroxyl and 3’ phosphate
May show specificity for where they attack,
Endonulceases cleave site within the molecule
Exonucleases degrade from end of molecule

Question 48

DNA methylation

Answer 48

During DNA replication only normal, non-methylated bases are used by dna polymerase
After dna replication of a duplex where both strands are methylated, the dna will be methylated on one strand only-hemimethylated
Hemimethylated dna is not cleaved by the restriction enzyme
Hemimethylated dna is the best substrate for the methylases so it quickly becomes fully methylated before the next round of dna replication
Totally unmethylated dna coming into a cell will be subject to methylation by the methylase and attacked by the nucleases so is normally degraded
Bacteria methylate their dna at sites that are recognised by the endonuclease to stop it being degraded- specific recognition sites are modified
Can be considered as a form of self recognition
Modification of dna by methylation within the restriction enzyme recognition site protects dna from degradation
C5 or N4 position of cytosine/ N6 position of adenine base.

Question 49

How are restriction enzymes named

Answer 49

First 3 letters is bacterial species of orgin (1 letter from genus and 2 from species, should be in italics)
Fourth letter-bacterial strain
Number (in roman numerals) differentiates between different enzymes in strain

Question 50

Type I REs

Answer 50

Asymmetric discontinuous recognition site
Methylate recognition site but cleave remotely
Multisubunit protein complex usually containing 2 restriction endonuclease subunits (Rease), 2 methyltransferase subunits (MTase) and one specificity subunit
ATP required for cleavage

Question 51

Type II REs

Answer 51

Symmetric recognition
Cleave and methylate within or close to recognition site
REase usually acts independently of MTase
Acts as monomer, dimer or even trimmers
ATP not required for cleavage
Can purify restriction enzyme and use on its own
Recognises palindromes

Question 52

Type III REs

Answer 52

Complex containing both REase subunit and MTase subunit
2 subunits
ATP required for cleavage
Asymmetric recognition site

Question 53

What is a palindromic sequence

Answer 53

Palindrome is a word that reads the same backwards or forwards
Mirror-like palindrome: Sequence reads the same for wards or backwards on a single strand of dna strand
Inverted repeat palindrome: sequence of top left strand reads left to right is the same as the sequence on the bottom strand reads right to left

Question 54

Compare types of dna cutting patterns

Answer 54

Sticky ends/ overhangs, useful to stick 2 pieces of dna together that are cut by same restriction endonuclease
Blunt ends – cut through arc of symmetry of palindrome.
Cut straight through middle
More difficult to ligiate
Can stick two ends together when cuts were made by different enzymes
Can’t stick blunt ends to sticky ends
Some restriction endonucleases cut at different sequences but make similar ends-can be easily ligated

Question 55

What are isochizomers

Answer 55

Enzymes that have the same recognition sequence
Not necessarily the same cleavage site or same sensitivity to methylation

Question 56

o Maxam-Gilbert

Answer 56

fragment the string at known positions and analyse size of fragments – label end of string, fragment at specific places, cuts specific for each base, measure length of fragments by electrophoresis

Question 57

o Illumina-solexa/ PacBio

Answer 57

synthesise a copy of the string and analyse the order in which the letters are added – sequence arrays, no electrophoresis, monitor as sequencing goes on

Question 58

o Oxford Nanopore

Answer 58

read the fragment directly – also uses arrays, no fragmentation needed, electrophoresis at nano scale

o Long reads are useful as there are repeated areas of DNA that make it hard to piece together small fragments. In long reads you can just see where repeats are