lab techniques: gel electrophoresis doug dluzen lab lecture 2 [email protected]
TRANSCRIPT
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 Methods
1. 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
