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Topics Genomic vs. Vector DNA Purifying plasmid DNA Restriction enzymes Basics of restriction mapping
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Molecular BiologyWorking with DNAWorking with DNA
TopicsTopics Genomic vs. Vector DNAGenomic vs. Vector DNA Purifying plasmid DNAPurifying plasmid DNA Restriction enzymesRestriction enzymes Basics of restriction mapping Basics of restriction mapping
DNADNA GenomicGenomic
Prokaryote vs. eukaryoteProkaryote vs. eukaryote Circular or linearCircular or linear One or more chromosomes One or more chromosomes
Extra-genomicExtra-genomic VectorsVectors PlasmidsPlasmids
Vectors Vs PlasmidsVectors Vs Plasmids Vector: Vector:
DNA vehicle that allows the cloning, DNA vehicle that allows the cloning, maintenance and amplification of a DNA maintenance and amplification of a DNA sequencesequence
PlasmidsPlasmids VirusVirus ChromosomesChromosomes
All plasmids are vectorsAll plasmids are vectors Not all vectors are plasmids Not all vectors are plasmids
PlasmidsPlasmids Small circular DNA molecules Small circular DNA molecules
maintained and amplified in maintained and amplified in eukaryotic or prokaryotic cellseukaryotic or prokaryotic cells Amplification in bacteriaAmplification in bacteria
Used as vector for cloning or Used as vector for cloning or expression of DNA of interestexpression of DNA of interest
Characteristics of plasmid vectorsCharacteristics of plasmid vectors
Restriction sites for Restriction sites for cloningcloning
Origin of replication Origin of replication (Ori)(Ori)
Selection markerSelection marker Genes conferring Genes conferring
resistance to antibioticsresistance to antibiotics
DNA IsolationDNA Isolation GoalsGoals
Isolation of DNA of interestIsolation of DNA of interest Chromosomal or plasmid?Chromosomal or plasmid?
Eliminate other componentsEliminate other components Chromosomal or plasmid DNA?Chromosomal or plasmid DNA? ProteinsProteins RNARNA ChemicalsChemicals
Salts, detergents, etc.Salts, detergents, etc.
DNA isolation DNA isolation (cont’d)(cont’d)
Cell lysisCell lysis Cell wall and membraneCell wall and membrane
EnzymaticEnzymatic ChemicalChemical MechanicalMechanical
Isolation of DNA of interestIsolation of DNA of interest Differential sedimentationDifferential sedimentation ChromatographyChromatography
Removing other componentsRemoving other components EnzymaticEnzymatic Differential sedimentationDifferential sedimentation ChromatographyChromatography
Plasmid DNA isolation by Plasmid DNA isolation by alkaline lysis (alkaline lysis (E.coliE.coli ) )
Solutions UsedSolutions Used Sol. I – Resuspension bufferSol. I – Resuspension buffer
Tris HCl – Buffer that protects nucleic Tris HCl – Buffer that protects nucleic acids acids
EDTA - Chelates Mg++, prevents EDTA - Chelates Mg++, prevents nucleases from working nucleases from working
Sol. II – Lysis solutionSol. II – Lysis solution NaOH - ^pH lyses cells, denatures DNA NaOH - ^pH lyses cells, denatures DNA SDS – Dissolves membranes, denatures SDS – Dissolves membranes, denatures
and binds proteins and binds proteins
Solutions Used Solutions Used (Cont’d)(Cont’d)
Sol. III- Potassium acetateSol. III- Potassium acetate Renaturation of DNARenaturation of DNA Precipitates SDSPrecipitates SDS Precipitates genomic DNA and proteinsPrecipitates genomic DNA and proteins
Isopropanol / Ethanol Isopropanol / Ethanol Precipitates nucleic acids (plasmid and ?) Precipitates nucleic acids (plasmid and ?) Salts remain soluble Salts remain soluble
TE-RNase - Tris & EDTA again; RNase??TE-RNase - Tris & EDTA again; RNase??
Quantification of DNAQuantification of DNA Determining Conc. of DNADetermining Conc. of DNA
A260 of 1.0 = 50µg/mL or 50ng/µLA260 of 1.0 = 50µg/mL or 50ng/µL
Determining Amount of DNADetermining Amount of DNA 1mL of a solution with an A260 of 1.0 contains 50µg DNA1mL of a solution with an A260 of 1.0 contains 50µg DNA 1µL of a solution with an A260 of 1.0 contains 50ng DNA1µL of a solution with an A260 of 1.0 contains 50ng DNA
Do not forget to account for the DILUTION FACTORDo not forget to account for the DILUTION FACTOR
Restriction enzymes Restriction enzymes EndonucleaseEndonuclease
Cleaves internal phosphodiester Cleaves internal phosphodiester linkages.linkages.
Recognize specific double stranded Recognize specific double stranded DNA sequencesDNA sequences
Different endonucleases recognize Different endonucleases recognize different sequencesdifferent sequences
Recognize palindromesRecognize palindromes
PalindromesPalindromes The same sequence is read in the The same sequence is read in the
5’ » 3’ direction on both strands5’ » 3’ direction on both strands
5’-GGATCC-3’3’-CCTAGG-5’
The same phosphodiester linkages The same phosphodiester linkages are cleaved on both strands!are cleaved on both strands!
5’-G
3’-C C T A G
G A T C C-3’
G-5’
Different ends are Different ends are generatedgenerated
5’-G
3’-C C T
G A
A G
T C C-3’
G-5’Blunt ends
Different ends are Different ends are generatedgenerated
5’ overhangs5’-G
3’-C C T A G
G A T C C-3’
G-5’
Different ends are Different ends are generatedgenerated
3’ overhangs3’-C
5’-G G A T C C-3’
C T A G G-5’
Compatibility of endsCompatibility of ends
OPO
P
Blunt ends
HOPOH
P
Compatible
Compatibility of endsCompatibility of endsOverhangs
HOPOH
P
HOPO
P
Incompatible
Compatibility of endsCompatibility of endsOverhangs
HOPOH
P
HOPO
P
Incompatible
Compatibility of endsCompatibility of ends
Overhangs
P-CTAGHOGATC-P
OH
Compatible
P-CTAGOGATC-P
O
Annealing
Compatibility of endsCompatibility of endsOverhangs
P-TCCAHOGATC-P
OH
Incompatible
P-TCCAHO
GATC-POH
Annealing
Mapping Mapping Restriction SitesRestriction Sites
Is the DNA digested?Is the DNA digested?
Must compare to an undigested control
D1ND D2
Is the digestion completeIs the digestion complete
Must compare toan undigested control
Partial
Complete
Partial DigestionPartial Digestion All the target molecules are not cut All the target molecules are not cut
at all the possible sites!at all the possible sites! Generates different intermediate Generates different intermediate
productsproducts
1 2 3
1 Product of a complete digestion
Product of a partial digestion (intermediate product)1 + 2
Product of a partial digestion (intermediate product)
2 + 3
Partial DigestionPartial Digestion
Complete Vs PartialComplete Vs Partial A complete digestion results in a A complete digestion results in a
stoechiometry of 1:1:1…stoechiometry of 1:1:1… The number of molecules of the The number of molecules of the
different fragments is the same!different fragments is the same!
The stoechiometry of a partial The stoechiometry of a partial digest is variable:digest is variable: Ex. 1:3:2…Ex. 1:3:2…
Ex.Ex.1 2 3
How many molecules of each of the fragments would be generated following a complete digest?
3; therefore a stoechiometry of 3:3:3 =1:1:13; therefore a stoechiometry of 3:3:3 =1:1:1
How many molecules of each of the fragments would be generated following a partial digest?
Assessing the Assessing the stoechiometry on an stoechiometry on an agarose gelagarose gel
BasisBasis The amount of ethidium bromide that binds The amount of ethidium bromide that binds
to DNA is proportional to the size of the DNAto DNA is proportional to the size of the DNA The amount of ethidium bromide that binds The amount of ethidium bromide that binds
to DNA is proportional to the amount of DNAto DNA is proportional to the amount of DNA With a complete digestion, the amount of With a complete digestion, the amount of
ethidium bromide that binds DNA is only a ethidium bromide that binds DNA is only a function of the size of the DNAfunction of the size of the DNA
Why?Why?
Ex.Ex.
Stoechiometry not respected
Stoechiometry respected
Stoechiometry not respected
Stoechiometry respected
ExamplesExamples
ExamplesExamples
11stst step: step: Determine the number of cutsDetermine the number of cuts Was the DNA digested? Was the DNA digested?
No- No mapping requiredNo- No mapping required YesYes
Is the digestion complete?Is the digestion complete? Is the digestion partial?Is the digestion partial?
Which products are intermediatesWhich products are intermediates How many times was the DNA cutHow many times was the DNA cut
The cut sites are in the vectorThe cut sites are in the vectorNo mapping requiredNo mapping required
The cut sites are in the insertThe cut sites are in the insertMapping requiredMapping required
How many restriction sites How many restriction sites are there?are there?
Linear DNA (ex. Human Linear DNA (ex. Human Chromosome)Chromosome) The number of cuts is equal to the The number of cuts is equal to the
number of fragments minus one.number of fragments minus one.
Circular DNA (ex. Plasmid)Circular DNA (ex. Plasmid) The number of cuts is equal to the The number of cuts is equal to the
number of fragments.number of fragments.
The cut sites are in the The cut sites are in the vector or the insert?vector or the insert?
B cuts 2 times in the vector and 0 times in the insertE cuts 1 times in the vector and 1 times in the insertP cuts 1 times in the vector and 2 times in the insert
insertB BE ?
S
Size of vector2.5kpb
P
2nd step: What are the 2nd step: What are the positions of the restriction positions of the restriction sites? sites?
Relative mapping is doneRelative mapping is done Restriction sites are mapped as a Restriction sites are mapped as a
function of a reference pointfunction of a reference point The position of the reference point The position of the reference point
must be knownmust be known
Must determine size of the Must determine size of the insert insert
1. Determine the size of the fragments resulting from a complete digestion2. Determine the total size of the plasmid (Sum of sizes)3. Determine the size of the insert (Size of plasmid - Size of vector)
insertB BE ?
S
Size of vector2.5kpb
P
Determining SizesDetermining Sizes
Thus: Size of plasmid 5KbpSize of insert 5000-2500=2500
Enz Distance (cm) Sizebp
P0.60.92.1
30001100900
E 0.31.9
45001000
Mapping Site PMapping Site P
Insert (2.5kbp)P
1.1kbpouP
1.1kbpP
Impossible since the second site must be in the insert!
Insert (2.5kbp)P
0.9kbpouP
0.9kbpP
Impossible since the second site must be in the insert!
Mapping Site PMapping Site P
Insert (2.5kbp)P
1.1kbpP
0.9kbpP
Insert (2.5kbp)P
0.9kbpP
1.1kbpP
Or
Determining Orientation Determining Orientation
Insert (2.5kbp)P1.0kbpE E
Determining OrientationDetermining Orientation
Insert (2.5kbp)P
1.1kbpP
0.9kbpP
Insert (2.5kbp)P
0.9kbpP
1.1kbpP
Or
1.0kbpE E
1.0kbpE E
Determining Orientation Determining Orientation (Cont’d)(Cont’d)