Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the...

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Electrophoresis and Blots

Transcript of Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the...

Page 1: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Electrophoresis and Blots

Page 2: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Principles recap

• Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis

• The resistance to movement is due to the sieving effect of the gel matrix

• The rate of migration is related to the size of the nucleic acid

Page 3: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Agarose versus acrylamide

• Acrylamide is a crosslinked synthetic polymer useful in separating DNA molecules of a few hundred bases in length– The crosslinking reaction is carried out within the molding

apparatus

– The final gel is transparent and usually used in a vertical apparatus

• Agarose is a polysaccharide similar to starch useful in separating DNA and RNA molecules of a few hundred to tens of thousands on bases long– A gel is created by boiling agarose into solution and allowing it to

cool within a molding apparatus

– The final gel is translucent and usually used in a horizontal apparatus

Page 4: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Restriction enzymes I

• Origin and native utility– Some restriction enzymes are used by bacteria to

protect themselves from invading DNA

– A “restriction-modification” system exists that marks bacterial DNA by methylating it on certain sequences

• A restriction enzyme specific for those sequences cannot digest DNA if it is methylated

– Invading viral DNA is unmarked and subject to cleavage at those sequences

Page 5: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Enzymes

• Restriction enzymes– Generally bacterial in origin

• But may come from lower eukaryotes, viruses or transposable elements

– Cleave DNA at short palindrome sites

• The palindromes may be 2 or more nucleotides long

• Unusual forms may recognize sequences up to 30 bases long

– These are used in site specific recombination events

• Cleavage usually results in a 5’ phosphate and a 3’ hydroxyl

• The resulting ends may be single stranded over a short region, or double stranded (blunt ends)

• The cleavage sites may be covalently linked back together by DNA ligase

Page 6: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Restriction enzymes III• A restriction enzyme with a 6 base recognition site

would produce on average a fragment size of 4096 bases in length when used to cut single copy DNA– The average fragment size can be determined for any

recognition site by the formula 4n where n is the number of bases in the recognition site

Size of recognition sequence

Average size of fragment

4 256

5 1024

6 4096

8 65536

Page 7: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Agarose gel electrophoresis

• Gels are loaded with samples of restricted DNA and electrophoresed for minutes to hours

• The voltage is turned off and the gel is stained with the intercalator ethidium bromide– DNA may be visualized in the

gel by illuminating it with ultraviolet light

– Linear relationship between mobility and the logarithm of the DNA size

Page 8: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Size determination• Size markers are commonly

used in electrophoresis experiments– These are duplex DNA

fragments of a known size– The migration distance of a

fragment may be compared to the migration of the size markers directly on the gel

– Alternatively the migration of a fragment may be determined by interpolation from a plot of the distance migrated by the size markers

• The distance migrated is related to the inverse log of the molecular weight of the DNA fragment

Migration versus size

100

1000

10000

100000

0 2 4 6 8

Distance migrated in cm

Nu

cle

oti

de

ba

se

pa

irs

Page 9: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Digesting human chromosomal DNA

• produces a range of fragment sizes due to cleavage of single copy DNA– Three billion base pairs cleaved by

a restriction enzyme recognizing 6 bases results in 750,000 fragments

• They appear as a background smear of DNA cleaved into all possible sizes by the restriction enzyme

• The fragments are of random size with an average length of >4096 bases

– Repetitive sequence can increase the average size, because usually a given restriction site will not be found within it

– If a restriction site is in a repetitive sequence, digestion will produce a large number of fragments of identical length

• This will appear as a band in the background smear of fragments

Page 10: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Southern Blot

• Once an agarose gel of digested chromosomal DNA has been stained, individual DNA sequences may be detected on the gel by hybridization– The DNA within the gel is denatured by exposing it to a

solution of sodium hydroxide– The DNA is then neutralized and transferred out of the gel

onto a membrane that binds DNA• This is a procedure known as blotting• It exposes the DNA to the surface so that it may hybridize to

complementary sequences

– The membrane bound DNA is then hybridized to a short specific sequence known as a probe

Page 11: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Probe• A probe is an oligo or poly nucleotide that

represents known DNA sequence – It is usually a sequence complementary to a

gene• But it only need be complementary to DNA

sequence within the chromosomal DNA itself

– It is either single stranded to begin with or is denatured to make it single stranded

– It is labeled with radioactive phosphorous or a fluorescent adduct

Page 12: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Hybridization• The membrane is

“prehybridized” with non-specific DNA to block non-specific binding sites on the membrane prior to hybridization

• It is then hybridized to the probe– The temperature and salt

conditions are controlled to insure optimal hybridization between the probe and the target sequences

– The single stranded probe finds its complement in the membrane bound DNA and forms a double helix

• Unbound probe is then washed off

Page 13: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

X-ray film exposure

• The membrane is exposed to X-ray film– Radioactive probes expose the

film whereever they hybridized on the blot due to emission of electrons (beta particles)

– Fluorescent probes catalyze a light yielding reaction using energy supplied in a reaction cocktail

• Development of the X-ray yields an autoradiogram– Bands on the film locate the

chromosomal restriction fragments complementary to the probe DNA

Page 14: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Overall

• Southern Blots– DNA is digested with restriction enzymes and

electrophoresed through an agarose gel– The contents of the gel are denatured in situ

and then transferred onto a membrane that binds DNA

– The DNA is hybridized to radioactive DNA complementary to the gene of interest

– Unhybridized DNA is washed away and the membrane exposed to X-ray film

• The technique can be adapted to identify any source of DNA containing a gene of interest or to RNA detection (northern blots)

Page 15: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Restriction fragment length polymorphism (RFLP)

• Chromosomal DNA from two people differs due to a random variations in sequence– There are millions of single nucleotide polymorphisms

between two unrelated individuals

• If one of these sequence variations results in the creation or destruction of a restriction site, then the fragment sizes of DNA will differ between these two people

• This can be detected by southern blots

Page 16: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Example I RFLP point mutation

• A gene sequence contains two Eco R1 sites separated by 4000 nucleotides

• A patient has suffered a mutation in one of his two homologous chromosomes such that a single nucleotide change has created a new EcoR1 site within the gene sequence– Now two fragments are created of 1500 and 2500 bases in length– The probe is complementary to sequence located within the 2500

base long fragment– Detection by southern blot of these two fragments will result in a

4000 base long fragment in normal DNA from one homologous chromosome and two fragments from the other homolog

Page 17: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

DNA from normal individual digested with Eco R1 and electrophoresed through agarose

DNA from mutant individual digested with Eco R1 and electrophoresed through agarose

Page 18: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Example II RFLP deletion mutation

How could these two patterns arise by deletion?

Page 19: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Example III Amplification

Page 20: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Other blots Northern

• Northern blots are mainly used to measure of the amount of a specific RNA in the cell

• The blot is prepared and hydridized like a southern blot

• There are a few minor technical alterations– The RNA is usually folded into a secondary structure so

it must be denatured before and during electrophoresis

• Bands on the autoradiogram are a measure of the presence of RNA from a particular gene– The more intense the band, the more RNA on the blot

Page 21: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Protein electrophoresis

• Proteins, unlike DNA, do not have a constant size to charge ratio– In an electric field, some will move to the positive and

some to the negative pole, and some will not move because they are neutral

– Native proteins may be put into gel systems and electrophoresed

• However native proteins are sometimes difficult to prepare without degradation

– An alternative to native protein gels forces all proteins to acquire the same size to charge ratio

Page 22: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

SDS-polyacrylamide gel electrophoresis

• A sample of protein, often freshly isolated and unpurified, is boiled in the detergent sodium dodecyl sulfate and beta-mercaptoethanol– The mercaptoethanol reduces disulfide bonds– The detergent disrupts secondary and tertiary structure– On the molecular level, proteins are stretched out and

coated with the detergent (which has a negative charge) by this treatment

– They will then migrate through a gel towards the positive pole at a rate proportional to their linear size

• Molecular weights with respect to size markers may then be determined

Page 23: Electrophoresis and Blots. Principles recap Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis The resistance.

Other blots II Western

• Western blots measure the amount of a specific protein in a cell

• This is a blot of an SDS- polyacrylamide gel onto which cellular protein is applied– The protein is denatured so it will enter

the gel as a stretched out random coil– The proteins separate by size– They are blotted onto a membrane

• Western blots are probed using antibodies– The antibodies are labeled. Those that

remain bound to their specific targets identify specific proteins in the cell

• The intensity of the signal reflects the level of expression

• It can also reveal mutations by anomalies of migration– A deletion might cause a shorter protein

for example