Molecular Biology Working with DNA

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)