Week 5: DNA analysis: Gel electrophoresis, Southern/ Northern Blotting, PCR

What does Gel electrophoresis do?

• separates fragments of DNA and RNA by size

• useful for analysing proteins

How does Gel electrophoresis work?

1. DNA is a negatively charged molecule at neutral pH

2. Therefore, in an electric field, will move towards the positive charge (anode)

3. Place a DNA preparation in a gel (molecular mesh - long polymer with side branches)

4. Apply an electric field to pull the molecules through

5. Small pieces of DNA move faster than big pieces of DNA.

What ingredients are needed for gel electrophoresis?

• buffer in solution (Tris= most widely used buffer)

• EDTA in solution → collating agent to take Mg and Ca ions

• Borate/ acetate also in solution → conduct electricity

• TBE = gel solution name

• Buffer permeates gel

• Mix DNA with a dye and glycerol (DNA to sink)

Solution: TBE→ TRIS, BORATE , EDTA

What kinds of gel can be used for electrophoresis?

1. Agarose→ low resolution

2. Acrylamide → high resolution

Where is agarose from?

Polysaccharide from seaweed

What is acrylamide and what problem is there with it?

• cross-linked polymer

• neurotoxin→ polymerises in nerve cells → loss of neurological function

How can you see the DNA in agarose gel?

• Use ethidium bromide

• A flat molecule that intercalates between the stacked bases of DNA, causing the dye to display an increased fluorescence compared to free dye (in solution)

• U.V. radiation at 254 nm is absorbed by the DNA and transmitted to the bound dye

• The energy is re-emitted at 590 nm in the red-orange region of the spectrum

• Is usually incorporated into the gel and running buffer

• The stain is visualized by irradiating with a UV light source (i.e. using a transiluminator)

• The usual sensitivity of detection is better than 0.1 ug of DNA.

What is wrong with ethidium bromide?

mutagen

What are size standards?

collection of marker fragments of known size

What are size standards used for?

To estimate the size of DNA fragments for agarose gel electrophoresis

Example of a size standard

HindIII digest of the lambda-phage genome

What are the 3 states of plasmids?

1. Supercoiled (the natural state)

2. Nicked (relaxed) circular→ break/ nick one strand- the other one pivots on the axis and ends up unwinding

3. Linear

Which plasmid state migrates the slowest?

linear→ less compact

What is nucleic acid hybridization?

• A property of single stranded DNA (and RNA)

• tendency to stick (or hybridize) to DNA (or RNA) molecules with a complementary sequence.

What does Northern/ Southern Blotting do?

• Rapidly detect very small amounts of specific DNA or RNA sequence in gels

• Southern = hybridisation of a DNA probe to a DNA sample

• Northern = hybridisation of a DNA probe to an RNA sample

How is Southern/ Northern blotting detected?

by hybridisation using a labeled (radioactive or fluorescent) probe - ie a piece of DNA of known sequence that is easily detected.

How does Southern Blotting Work?

1. DNA fragments cut with restriction nuclease

2. Fragments separated by agarose gel electrophoresis

3. Separated DNA fragments blotted onto nylon filters

4. Labelled DNA probe hybridized to separated DNA

5. Labelled DNA probe hybridized to complementary DNA bands visualised by autoradiography

What was used before nylon filters?

nitrocellulose was used → DNA naturally sticks and binds→ not used now→ explosive

What does the stringency (temperature at which the probe is washed off of the membrane) determine?

how similar the probe has to be to it’s target to hybridise

Basic science applications of Southern Blotting

• Insertion of genes: have you made the change you want to make

• Finding gene homologues in other organisms

• Looking at genetic variation in populations→ finding variable number of tandem repeats

• Finding clones in a DNA library - colony blots → cloning blots into bacterial plasmids → bacteria take up DNA if treated with CaCl2

Biomedicine/diagnostics/forensics applications of S. Blotting

• Detection of specific DNA sequences/polymorphisms (but note that there are often alternatives based on PCR)

• DNA fingerprinting

• Fluorescence in situ hybridisation (FISH) - uses in prenatal screening (for example) – FISH is not strictly Southern Blotting, but it relies on the same hybridisation idea.

What can Northern Blotting do specifically?

• Northern blotting tells you which genes are being transcribed in a particular organism / tissue under a specific set of circumstances

• Used for monitoring levels of expression of specific genes.

Difference between Southern Blotting and Northern Blotting?

• RNA is ss, DNA is ds→ single stranded nucleic acid molecules fold and some parts can become ds so won’t exactly run according to size

• Add chemicals to cause DNA + RNA→ ss

• Soak gel in formaldehyde and formamide + heat it up

• Northern Blotting hard to do

• RNA hard to extract → breaks up easily

• formamide + formaldehyde are not nice chemicals

• blotting is arduous and lengthy

What can Northern Blotting be replaced by?

Expression arrays

What are expression arrays

• looks at all possible genes in the genome

• Variations using “gene chips” are now a more way of identifying genes involved in particular physiological responses

• comparative hybridisation

How do expression arrays work?

• DNA sequences immobilised on a glass slide→ each dot contains a sequence from a different gene

• Take RNA from a particular tissue → cDNA using reverse transcriptase and make that fluorescent using a nucleotide labelled with a fluorescent dye

• Wash the dye over the whole expression array

• Only the mRNA present in the original tissue are going to hybridise to the gene sequences in these dots

• RNA present → gene switched on

• Take 2 tissues → one red fluorescent, one green

• Wash dye over

• Orange → gene present in same amount

• Green/ red - more present in one tissue than in the other

• compare in high detail the expression levels of genes in different tissues/ at different times/ under different conditions

What does PCR do?

Method for making millions/billions of copies of specific, short segments of DNA using very small amounts of starting template

What are the products of PCR used for/ in?

DNA sequencing, identification of infectious agents, other forms of analysis, and DNA manipulation

Template DNA

The source DNA to be copied / amplified

Primers

Short pieces of single-stranded DNA that specify the region of the template to be copied / amplified.

Taq polymerase

A heat-stable DNA polymerase obtained from the thermophilic bacteria Thermus aquaticus (or similar)

Oligonucleotides

short, single stranded sections of DNA of known sequence that have almost always been synthesised by a machine → which makes DNA in the 3’ to 5’

Denaturation

• The separation of strands in a double-stranded DNA molecule by heating

• Temperature depends on sequence

• ~95 C

Annealing

• The hybridization of primers to template DNA by cooling to an appropriate temperature

• 50-65 C (~55 C)

Extension

• The synthesis of double-stranded DNA from single-stranded DNA with an annealed primer

• ~72 C

dNTPs

deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP)

Thermal cycler

A heater block that rapidly heats and cools sample tubes

DNA Replication reminder

• Always in 5’ to 3’ direction!!!!!

• Requires:

  1. Single-stranded template with double stranded section (> 4 bases) and free 3’ end

  2. dNTPs

  3. DNA polymerase

PCR Reaction Ingredients

• Template DNA – from almost any type of biological material

• 4 types of nucleotides (aNTPs)

• 2 primers

• Thermostable (e.g. Taq) DNA polymerase

• Buffer solution – providing a suitable chemical environment for polymerase activity→ Tris (maintain pH ~9)

• MgCl2

Issue with PCR for ancient DNA

• can accidentally amplify your own DNA → contamination

• dust as skin cells

How many cycles of PCR will usually produce enough amplified DNA for most forms of genetic analysis?

• 35 cycles

• 34,359,738,368 from one starting molecule

What happens to the long bits/ Why does it end up that the majority of your products are only (after 30/ 35 rounds) the distance between the two primers?

• Over successive rounds more and more of the newly synthesized strands will be of length equal to the distance between the two primers

• This is because over successive rounds more of the single stranded templates will themselves be the products of previous rounds of amplification

• As the PCR process progresses, more of the templates are the products

Problems with PCR- Contamination

PCR is extraordinarily sensitive, especially when one performs many cycles. Just a few contaminating molecules can give misleading results (e.g. from the scientists own skin)- dust is skin cells

Problems with PCR - Inappropriate Priming

• PCR primers can initiate DNA replication even if they don’t match a piece of template DNA perfectly (especially if they do match the template DNA at the 3’ end of the primer)

• This can produce additional DNA products, or sometimes, completely the wrong products

• The lower the temperature, the more likely the primer will anneal and extend somewhere else.

Problems with PCR - Taq Errors

Taq polymerase mediated DNA synthesis is error prone (~0.5 % errors= an error in 1/ 200 bases). i.e. Taq will sometimes put the wrong base in the DNA chain it is synthesizing.

Problems with PCR- Optimisation

Factors such as Magnesium concentration, primer concentration and annealing temperature often need to be optimized before a PCR can work efficiently.

Problems with PCR- Difficult sequences

some DNA sequences are difficult to amplify → GC rich sequences.

Why are thermostable polymerases important in PCR?

• not degraded during the heating stage of the reaction

• PCR without thermostable polymerase→ heated to denature, added more polymerase→ heated, then added…

Why are primers especially useful in PCR?

can be modified to introduce specific changes into the amplified DNA sequence = site-directed mutagenesis

Main considerations for good primer design

• Annealing temperature matching

• Length and GC content

• Primer dimers

• Hairpins

• Repeat sequences / unique sequences

• GC clamp

How to design primers

• The primer should on a 5’- 3’ direction be pointing into the region you want to amplify

• Top going left, bottom going right

• One primer needs to go right and the other left to focus/ amplify the region

• Always write primer sequences as ss, 5’→ 3’

• The upper primer will be that sequence written in the 5’→ 3’ direction

• The lower primer will be antiparallel → sequence written in the 5’→ 3’ direction

Annealing temperature matching (primer design)

• During the annealing stage of PCR, if the temperature goes below the annealing temperature of the primer, then it will anneal to non-target locations

• It is therefore important for the 2 primers to have similar annealing temperatures

Length and GC content (primer design)

• GC pairs stick more strongly than AT pairs

• The more G and C a primer has, the higher the annealing temperature

• The longer a primer is, the higher the annealing temperature

How can we control the annealing temp?

by making the primer longer or shorter, and by choosing primers with higher or lower GC content

Primer Dimers

• Primer dimers primer can lead to the synthesis of random products - polymerase loves ss with short ds section

• Should avoid primers that hybridise to themselves or to one another at their 3’ ends

Hairpins (primer design)

• Primers that form hairpin structures can be less efficient

• Hairpin structures with a 5’ overhangs (as above) can lead to further extension from the 3’ end, creating a nonsense primer

• 3’ overhang is not a problem- not a substrate for polymerase

Repeat sequences / unique sequences (primer design)

• Should avoid primers that anneal to multiple locations throughout the genome, and many different PCR products will be produced

• Better to target unique sequences (this is possibly by searching genome sequence databases)

GC Clamps (primer design)

• Many people argue that it is important to have at least one C or G in the last 5 bases of a primer

• believed to make it stickier/ easier to hybridise

• BUT potentially a bad thing → more likely to hybridise/ stick elsewhere