Basic Principles and Applications of Electrophoresis

Stephen K.W. Tsui

Department of Biochemistry

Order Form for Electrophoresis Kits and Reagents

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Theory of Electrophoresis

The movement of a charged molecule subjected to an electric field is represented by the following equation:

V =Eqf

V: the velocity of the molecule

E: the electric field in volts/cm

q: the net charge on the molecule

f: frictional coefficient, which depend on the mass and shape of the molecule

Applications of Gel Electrophoresis

• Southern blot is produced when DNA on a nitrocellulose blot is hybridized with a DNA probe.

• Northern blots are generated when RNA is hybridized with a complementary DNA probe produced by the reverse transcription of messenger RNA.

• A slightly different but related technique, known as a Western blot, involves separating proteins by gel electrophoresis and probing with labeled antibodies for specific proteins.

Southern Blot

Northern

Blot

Western

Blot

Macromolecules on the blot

DNA RNA Protein

Probe Labeled DNA Labeled DNA Labeled antibodies

Source of labels Radioactively or fluorescent

labeled deoxynucleotide

Radioactively or fluorescent

labeled deoxynucleotide

Radioactively or fluorescent

labeled amino acids

Blotting Techniques

Tissue distribution Tissue distribution of Messenger of Messenger RNA RevealedRNA Revealed by Northern by Northern BlotBlot

1 2 3 4 5 6 7 81 2 3 4 5 6 7 8

9 : spleen10: thymus11: prostate12: testis13: ovary14: small intestine15: colon16: leukocyte

1: heart2: brain3: placenta4: lung5: liver6: skeletal muscle7: kidney8: pancreas

9 10 11 12 13 14 15 16

A Protein can be Specifically Recognized A Protein can be Specifically Recognized by an Antibody in a Western Blotby an Antibody in a Western Blot

M 1 2 3 4M 1 2 3 4 M 1 2 3 4M 1 2 3 4

Coomassie BlueCoomassie Blue Dye Dye Stained Stained protein gelprotein gel Western blotWestern blot

Gel Electrophoresis of DNA

 

Agarose slab gel submerged in buffer

Wells for sample loading

Cathode (-)

Direction for DNA migration Anode (+)

Agarose is a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution. When an electric field is applied to an agarose gel in the presence of a buffer solution which will conduct electricity, DNA fragments move through the gel towards the positive electrode (DNA is highly negatively charged) at a rate which is dependent on its size and shape.

Gel Electrophoresis of DNA

For linear DNA molecules, they have uniform shape and charge to mass ratio. The electrophoretic mobility of the DNA molecule is influenced primarily by the molecular size: The larger molecules are retarded by the molecular sieving effect of the gel, and the small molecules have greater mobility.

Gel Electrophoresis of DNA

• The DNA can be stained by the inclusion of ethidium bromide in the gel, or by soaking the gel in a solution of ethidium bromide after electrophoresis. The DNA shows up as an orange band on illumination by UV light. Alternatively, methylene blue can be used to stain DNA.

• Gels composed of polyacrylamide can separate DNA molecules that differ in length by only one nucleotide and are used to determine the base sequence of DNA. Agarose gels are used to separate DNA fragments that have larger size differences.

Procedures of DNA Fingerprinting

• In order to detect specific sequences, DNA is usually transferred to a solid support, such as a sheet of nitrocellulose or nylon paper.

• The paper is treated with an alkaline solution to denature DNA, that is, separate the two strands of each double helix.

• The single-stranded DNA can be hybridized with a probe, and the regions on the nitrocellulose blot containing DNA that base-pairs with the probe can be identified.

Procedures of DNA Fingerprinting

DNA Polymorphisms

• Polymorphisms are variations in DNA sequences. There may be millions of different polymorphisms in the human DNA.

• Polymorphisms in the human DNA serve as the basis for the diagnosis of diseases and the identity of individuals.

Detection of Polymorphism Restriction Fragment Length Polymorphisms

• Occasionally, a point mutation occurs in a recognition site for a restriction enzyme. The enzyme, therefore, can cut at other recognition sites but not at the site of the mutation. Consequently, the restriction fragment produced by the enzyme is larger for a person with the mutation than for a normal person.

• Mutations can also create restriction sites that are not present in the normal gene. In this case, restriction fragments will be smaller for the person with the mutation than for the normal individual. These variations in the length of restriction fragments are known as restriction fragment length polymorphisms (RFLPs).

Application of DNA Fingerprinting Mutation Detection

Highly Variable Regions

• Human DNA contains many sequences that are repeated in tandem a variable number of times at certain loci in the genome. These regions are called hypervariable regions because they contain a variable number of tandem repeats (VNTR).

Digestion with restriction enzymes that recognize sites which flank the VNTR region produces fragments containing these loci, which differ in size from one individual to another, depending on the number of repeats that are present. Probes used to identify these restriction fragments bind to or near the sequence that is repeated.

Detection of Highly Variable Regions

Application of DNA Fingerprinting Forensic Analysis

This restriction fragment technique has been called "DNA fingerprinting" and is gaining widespread use in forensic analysis. Family relationships can be determined by this method, and it can be used to convict suspects in criminal cases. Individuals who are closely related genetically will have restriction fragment pattern that are more similar than those who are more distantly related.

Other Applications of DNA Fingerprinting

• Parentage test

• Endangered species or Chinese herbs identification

Animation 1: Southern Blotting

http://www.dnalc.org/resources/BiologyAnimationLibrary.htm

Animation 2: DNA Detective

http://www.dnalc.org/resources/BiologyAnimationLibrary.htm

Online Courses: DNA from the Beginning

http://www.dnaftb.org/dnaftb/

Download Illustrations: Human Molecular Genetics

http://www.bios.co.uk/illustrations.asp

http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/

gel.html

Good Website: Gel Electrophoresis

Workshop

Agarose Gel Electrophoresis

Department of Biochemistry

(2001-2002)

Restriction enzymes - enzymes isolated from bacteria that cut DNA at specific sites(restriction sites)

EcoRI - 5'- G A A T T C -3‘

3'- C T T A A G -5'

Properties of DNA-

Double helixBuilding block(dA, dC, dG and dT)negatively charged at neutral pHAT and GC complementary pairing

Baterial plasmid DNA

Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome.

They:

are small (a few thousand base pairs)

usually carry only one or a few genes

are circular

have a single origin of replication

+

BglI 2166

BglI 472

EcoRI 701

Plasmid DNA for digestion

– pBluescript II SK+

Agarose

A linear polymer extracted from seaweed

Migration of DNA in agarose dependent on four factors

- molecular size of the DNA- agarose concentration- conformation of the DNA- applied current

wells

Anode(+)

Cathode(-)

1.5% agarosegel stained with methylene blue

DNA fragments of different sizes

Preparation of plasmid DNA

Restriction enzyme digestion

» Agarose gel casting

» DNA sample loading

» electrophoresis

Methylene blue staining

http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html

http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html

Agarose gel electrophoresis unit

Gel casting unit and comb

Electrophoresis tank

Plugs and wire

Seal both ends of the gel casting unit with tape

Agarose gel casting unitStep 4

Gel casting unit

Comb

Tape

Preparation of 1.5% agarose gelStep 5

http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html

Electrophoresis(5V/cm)

Sample loading, wash syringe with 1X TBE buffer between successive loading

Step 10

http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html

Wells

Bromophenol blue

Xylene cyanol FF

Tracking dye

Methylene blue staining

» to visualize the DNA fragments, stain agarose gel overnight with 1X methylene blue staining solution

» safe alternative for DNA staining

» easy available

» non-carcinogenic

DNA fragments

Base pairs distance migrated(mm)

A B C D M

316 47

656 38

1808 231634 25

DNA fragments of different sizes

DNA fragments of known sizes

Calibration curve for DNA size determination

http://www.pangloss.com/seidel/Protocols/webmap.html

Size determination of the candidate DNA fragments

http://www.pangloss.com/seidel/Protocols/webmap.html

Workshop

DNA Fingerprinting

&

Agarose Gel Electrophoresis

Department of BiochemistryCUHK

(TDC2003)

nucleus

Human genome ~ 3 billion base pairs.

5 % of the genome are protein coding sequence (30,000 genes).

95% non-coding DNA.

20-30% are repetitive.

Animal cell

Repetitive DNA

Tandemly repeated sequences (~10% of genome)

Interspersed elements(~15-20% of the genome)

Satellite DNA Minisatellites / VNTRs SINES eg. Alu (3-6%)

LINES eg L1(1-2%)

(J.C.S. Fowler et al.)

VNTRs : Variable number tandem repeats

Rich source of genetic variation

Identical twins

You and me

0.1 % vary person to person

Each of us have unique DNA fingerprint / personal barcode.

Two kinds of DNA polymorphic regions (regions of different)

Sequence polymorphism Length polymorphism

…ACGTAGCAGCAGCG…

…TGCATCGTCGTCGC…

A, T

Simple substitution of one or two bases in the gene

T, A

Simple variations in the physical length of the DNA(eg. VNTRs)

DNA fingerprint/Length polymorphism in a single locus

M P

AT

AT

AT

AT

AT

AT

M P

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

M P

AT

AT

AT

AT

AT

AT

AT

Person 1Homozygous

Person 2Heterozygous

Person 3Heterozygous

AT VNTR repeating unit

M Maternal chromosome copyP Paternal chromosome copy

Variable number tandem repeats (VNTRs)

VNTRs are not distributed evenly across human population.

Each of allele occurs at a certain frequency in a population.

Each locus usually has approximately 30 different alleles.

Frequency of allele A at one locus = 0.1 (10%) Frequency of allele B at second locus = 0.05 (5%) Frequency of the two alleles of the loci occur together = 0.1 X 0.05 (DNA profile frequency) = 0.005

or 1 in 200

M P

ATATAT

ATATATATATAT

ATATAT

Locus X Locus Y

Locus Z  X Y Z

Father 10, 14 18, 24 8, 9

Mother 12, 20 22, 22 8, 15

Son 10, 20 22, 24 8, 8

Daughter 12, 14 22, 22 26, 9

Chromosome A

Chromosome B

DNA profile frequency for 3 loci: ~1 in 3 million

Application of DNA fingerprinting

Paternity and maternity test.

Criminal identification and forensics.

Personal identification.

Source of human DNA for fingerprinting

Whole blood

Buccal epithilial cells

Hair follicles

Semen

                 

Double stranded target DNA

Two DNA targets available for PCR

PCR amplification

Cycle(35 – 40 cycles)

DNA denatured95oC

Step 1 : Denaturation

Primers bind to target DNA~55oC

Step 2 : Primer Annealing

Double stranded DNA duplicated72oC

Step 3 : DNA Extension

Amount of amplified DNA = 2n x Cwhere n = number of PCR cycles; and C = the initial number of copies of DNA template present in the tube.

So, you will get 1,048,576 copies of DNA after 20 cycles of PCR reaction even you start with only one copy of DNA template initially

Polymerase chain reaction

Maternal tandem repeat fragment

Paternal tandem repeat fragment

Ethidium bromide stained agarose gel

Electrical wire1ml syringe filtted with tip

10 X TBE concentrate

Electrophoresis unit

Gel casting unit

Home made gel electrophoresis kit

Agarose

A linear polymer extracted from seaweed

Migration of DNA in agarose dependent on four factors

- molecular size of the DNA- agarose concentration- conformation of the DNA- applied current

Seal both ends of the gel casting unit with tape

Agarose gel casting unit

Gel casting unit

Comb

Tape

Preparation of 1.5% agarose gel

Wells

Electrophoresis(5V/cm)

Sample loading

DNA is negatively charged

Power supply

Nine 9V batteries connected in series

Three 24V adaptor connected in series

Wells

Bromophenol blue

Xylene cyanol FF

Tracking dye

1cm

Cathode (-)

Anode (+)

Methylene blue staining

» to visualize the DNA fragments, stain agarose gel overnight with 1X methylene blue staining solution and destain in distilled water for 3 – 4 hours

» non-toxic

» easy available

» non-carcinogenic

Lane MK: DNA marker Lane M: Mother Lane F: Father Lane D1: Daughter 1 Lane D2: Daughter 2 Lane S1: Son 1 Lane S2: Son 2

2100 bp

1700 bp

1100 bp

850 bp

600 bp

250 bp

MK M F D1 D2 S1 S2

Results

(Biologic child / adopted child / stepchild)

Calibration curve for DNA size determination

Size determination of the candidate DNA fragments

online size determination

THE END