Lab Techniques: Gel electrophoresis

Lab Techniques: Gel electrophoresis

Doug DluzenLab Lecture 2

ddluzen@hmc.psu.edu

What goes in a Gel?• Agarose (1%)

– Can range from 0.5% to 2.0% depending on fragment size• TAE Buffer

– Mixture of Tris base, acetic acid, and EDTA– Commonly made as a 50X Stock and diluted to 1X Stock with Water – At 1x general concentrations are as follows:

• 40 mM Tris• 20 mM acetic acid• 1 mM EDTA

• Water• Ethidium Bromide

Ethidium Bromide• Hazardous!!!

– Possible carcinogenic properties– Handle with care

• Used as a fluorescent tag to visualize DNA and RNA– Expose gel with UV light and DNA bands will glow and can be

visualized– Incorporate into DNA can induce up to 20-fold intensity of

flourescence– Intercalates between DNA and RNA base pairs

http://course1.winona.edu/sberg/ILLUST/eth-br.JPGhttps://web3.unt.edu/riskman/Images/EtBr_CAS_1239-45-8.bmp

How to Make a Gel• 1. Dilute 50x stock reagent TAE buffer into a 1x using water• Add 1% weight to volume dry agarose

– i.e. 0.5 grams agarose into 50 mL 1x TAE buffer solution• Heat up in microwave until all agarose dissolved• Cool down enough to add EtBr (generally 4-5 uL)• Pour gel (watch for air bubbles!) and allow to solidify (~20

minutes)• Add DNA/RNA samples into lanes

– Mixed with Loading Dye to visualize lanes– Be sure to include controls and DNA size ladder

Gel Electrophoresis• One indirect method of rapidly analyzing and

comparing genomes is gel electrophoresis• This technique uses a gel as a molecular sieve to

separate nucleic acids or proteins by size, electrical charge, and other properties

• A current is applied that causes charged molecules to move through the gel

• Molecules are sorted into “bands” by their size

Adopted from Dr. Sairam Lecture Slides

Pulsed field gel electrophoresis

Adopted from Dr. Sairam Lecture Slides

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/dsDNA.jpg

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Cathode Anode

Wells

Gel

Shortermolecules

TECHNIQUE

RESULTS

1

2

Adopted from Dr. Sairam Lecture Slides

Analyzing DNA

Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites

• A restriction enzyme usually makes many cuts, yielding restriction fragments

• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends.”

Adopted from Dr. Sairam Lecture Slides

Restriction Digest Sites

Adopted from Dr. Sairam Lecture Slides

DNA Ligase• Sticky ends can bond with complementary sticky

ends of other fragments• DNA ligase is an enzyme that seals the bonds

between restriction fragments

Adopted from Dr. Sairam Lecture Slides

Recombinant DNA molecule

One possible combinationDNA ligaseseals strands

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

Restriction enzymecuts sugar-phosphatebackbones.

Restriction site

DNA5

5

5

5

5

5

5

5

55

5

5

55

5

5

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

2

3

1

Sticky end

GAATTCCTTAAG

CTTAAG AATTC

G

GGAATTC

CTTAA

GG

GG

AATT CAATT CC TTAA C TTAA

Adopted from Dr. Sairam Lecture Slides

Using Restriction Enzymes • In restriction fragment analysis, DNA fragments

produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

• Restriction fragment analysis can be used to compare two different DNA molecules, such as two alleles for a gene if the nucleotide difference alters a restriction site

• Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms)

Adopted from Dr. Sairam Lecture Slides

Normal -globin allele

Sickle-cell mutant -globin allele

Largefragment

Normalallele

Sickle-cellallele

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal andsickle-cell alleles of the -globin gene

(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles

201 bp175 bp

376 bp

Large fragment

Large fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI

RFLP Analysis

Adopted from Dr. Sairam Lecture Slides

Restriction Digests• Need to check if PCR fragment, generally

cloned into a useful reporter, has the correct orientation– DNA inserts can ligate into a plasmid in two

directions

Detection of Specific DNA and Protein Sequences - Blotting

• Whether you are analyzing DNA, RNA, or protein the underlying principle is the same

• Use of a probe that signals presence/absence of a gene or protein of interest

• Labeled (chemically, radioactively, etc.)probe is used to visualize your target

• A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization

• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

Southern Blotting

Adopted from Dr. Sairam Lecture Slides

Southern Blotting

Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns

Southern Blotting

A southern blot can distinguish:1. The presence of a particular gene of interest2. Number of copies of that gene3. Genomic rearrangements4. Mutations of restriction digest sites

-Southern blots are very sensitive

Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns

Northern Blotting• Northern blotting combines gel electrophoresis of

mRNA followed by hybridization with a probe on a membrane

• Identification of mRNA at a particular developmental stage suggests protein function at that stage

Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northernsAdopted from Dr. Sairam Lecture Slides

Northern Blotting• Same principle as southern blotting, except

RNA is measured as opposed to DNA– RNA can also bind to nitrocellulose membrane– Uses formaldehyde as a denaturing reagent

• Used to identify tissue and temporal expression of a particular gene– Sensitive– Used to measure expression levels of particular

mRNA

Western Blotting• Powerful tool to detect presence and expression

levels of a particular protein– Use of an antibody – specific protein molecule will bind

to specific protein sequence on the protein of interest• This specific protein sequence is called an epitope

• As with northern and southern blotting, proteins are sorted by molecular weight, transferred to a membrane, and probed– Protein presence, expression, and quantity can be

measured

Western Blotting - Principles

Adopted from GE Healthcare: Western Blotting Principles

Western Blotting Methods1. Electrophoresis – Denaturing Gel

Adopted from GE Healthcare: Western Blotting Principles

Western Blot Methods2. Transfer from gel to membrane

Adopted from GE Healthcare: Western Blotting Principles

Western Blot Methods• Once protein transferred to membrane– Incubate in protein buffer (generally 5% milk

solution) to bind all regions of blot not bound by transferred protein

• Incubate with primary and secondary antibodies

• Visualize!

And now for something completely different…

Browsing the Genome Browsers• NCBI - http://www.ncbi.nlm.nih.gov/

• Ensembl - http://useast.ensembl.org/index.html

• University of California Santa Cruz - http://genome.ucsc.edu/

• Pubmed - http://www.ncbi.nlm.nih.gov/pubmed/

NCBI

BLAST!

Ensembl

Ensembl – THE MANY USES

I see U see UCSC

UCSC

Other Useful Odds and Ends• HapMap Project – encyclopedia of human and

mouse SNP variation and genotype frequencies www.hapmap.org

• TargetScan – microRNA prediction • Pubmed – uses NCBI database for literature

searches, protein and nucleotide sequences

Thank you! Questions?

ddluzen@hmc.psu.edu