Lecture Four - Restriction Mapping/Electrophoresis

Motion of charged particles in an External Electric Field

Negative

Ethidium Bromide

An intercalating agent used in Labelling DNA fragments on electrophoresis gel.q

The DNA was radioactively labelled and had to be assayed.

Radioctive Assays are slow and cumbersome.

It binds non-specifically to the DNA and fluoresces under UV light.

• Polyacrylamide
• Agarose

Agarose Gel

They form sieve-like pores that Retard molecules by size.

It has smaller pores

• Better Resolving Power
• Better at Seperating Smaller Molecules

• Difficult to prepare (polymerize)
• MonoAcrylamide is considered a Neurotoxin

• Non-toxic
• Horizontal Configuration

Agarose (50bp-30kb)

• At low voltages Migration is proportional to potential difference.
• At high voltages motion is more (exponential?).

5-8V/cm

Change the Agarose concentration

For smaller DNA fragments

Smaller pores -> Less Range of suitable sizes

• 30kb. DNA larger than this does not seperate.
• Use Sequencing

• In order of least migration:
• 1. Linear DNA
• 2. Nicked Plasmids
• 3. Supercoiled Plasmids

Two bands very close together

• The Linear DNA
• The Nicked DNA

Usually some damage will occur during extraction resulting in Nicking or Complete breaking of the plasmid.

A plasmid with a single stand of its ds-DNA broken which causes it to relax (from it's supercoiled form) into a more circular shape.

DNA Gyrase

Supercoiled

Linear Plasmids

It slows it down

It binds and reduces the negative charge of the DNA molecule.

Being a bulky molecule it alters DNA's stiffness and strength.

• It's charge
• It's Stiffness
• It's strength

35-38°C

*~98°C

Agarose with a much lower melting point than normal.

30-45°C

• Altering DNA without extraction (melt and modify)
• Bacterial Transformation can occur in the gel
• Low Melting Point Agarose can stabillize DNA

Because purification always results in some loss of DNA

No migration will occur due to insufficient ionic strength

The gel could overheat

• First, agarose is dissolved in a buffer
• The gel is then solidified and placed in a buffer chamber
• The gel is now ready for loading

• To see if the well is punctured
• Dissolved sucrose increases density meaning DNA sinks to the bottom
• Move predictably in an electric field (like a rough placemarker for DNA)

• Price
• Preference
• Size of DNA
• Potential Obscuring

• It binds nonspecifically to the DNA and absorbs UV light.
• The UV light is then re-emitted as red-orange light

It makes it more fluorescent

• It is susceptible to contamination
• Gloves must be warn (mutagenic)
• Best added after gel is run

An intercalating agent used in detecting DNA for electrophoresis

• It is expensive
• It is packaged with DMSO which will carry it across skin

• It binds to DNA
• It is non-mutagenic

Because it interacts directly, it may cause mutations or damage.

A standard curve produced by some standard form(s) of DNA

• Known DNA sequences digested by known enzymes (e.g Lambda + HindIII)
• Commercial Preparations (2-log, 1kb plus etc)

No, a new curve must be made for every gel

Usually the leading edge, but it doesn't matter so long as you are consistent

Roughly 5-6

Electrophesis using an alternating electric field

HeteroPolymers consisting of two types of Galactose molecules

• D-Galactose
• 3,6-anyhdro L-Galactose

• Electrical Current/Voltage
• DNA Size
• Concentration of Agarose
• DNA Conformation
• Ethidium Bromide
• Agarose Type
• Buffer used

V=IR

Take the recirocal of log the log of the base pairs (1/(log₁₀(bp))

0.5% Agarose

1.2% Agarose

2.0% Agarose

It has been modified by Hyroxyethylation

• Provide ions to carry current
• Maintenance of pH

• Tris-Acetate + EDTA (pH8.0)
• Tris-Borate + EDTA (pH8.0)

At high concentrations

0.5x - 1.0x

Tris-Acetate and EDTA, it is an electrophoresis buffer.

Tris-Borate & EDTA, it is an Electrophoresis buffer

TBE

TAE

TAE

TBE

TAE

• High Buffering Capacity
• Re-usuable

TBE

10ng

10 mg/mL

0.5ug/mL after dissolving Agarose

Complex Secondary strctures can give strange migrational patters (i.e it misbehaves)

• Using Agarose Gels Containing Formaldehyde
• (Or) Urea Containing Polyacrylamide Gels

• Immobillize Cells in Low Melting Point Agarose
• Lyse cells
• Digest DNA with a Rare Cutter

It can be melted and the cells whithin further modifed

• Detergent
• RNAase
• Proteinase K

Often NotI (8bp recognition sequence)

The gel (and buffer) surrounded by lots of evenly spaces electrodes (often in a hexagonal arrangement)

Bent lanes

• The alternating field causes the DNA to distort and snake through the pores of the gel reorienting with every "pulse"
• Larger molecules take longer to reorient

• Heat the RNA in it's buffer then add Formamide and Ethidium Bromide
• Next Make a solution of Agarose and Formaldehyde

1-1.5%

2.2M

Better resolution (diference of 1nt)

• The reaction is Initiated by free radicals initiated by Ammonium Persulfate.
• Catalysed by TEMED

• Restriction
• Separation

Gel Electrophoresis

• Systematics
• Determining Information about a Clone
• Determining orientation of DNA insterted into a plasmid

Looking at Relatedness

• Locating a particular fragment
• Size Determination
• Locating restriction sites for future reference

• Sequencing is limited to 600bp
• A starting point and comlimentary nucleotides must be added
• It takes time

~1Kb

9-50kb