Molecular Biology Working with DNA. Topics Genomic vs. Vector DNA Purifying plasmid DNA Restriction enzymes Restriction maps.

Molecular Biology

Working with DNAWorking with DNA

TopicsTopics

Genomic vs. Vector DNAGenomic vs. Vector DNA Purifying plasmid DNAPurifying plasmid DNA Restriction enzymesRestriction enzymes Restriction maps Restriction maps

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 maintenance and amplification of a DNA sequenceDNA sequence

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 Recognize palindrome sequencespalindrome sequences

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


5’ overhangs5’-G

3’-C C T A G

G A T C C-3’

G-5’


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


Overhangs

HOPOH

P

HOPO

P

Incompatible


Overhangs

P-CTAGHOGATC-P

OH

Compatible

P-CTAGOGATC-P

O

Annealing


Overhangs

P-TCCAHOGATC-P

OH

Incompatible

P-TCCAHO

GATC-POH

Annealing

Restriction MapsRestriction Maps

Restriction mapsRestriction maps

Determining the positions of Determining the positions of restriction enzyme sitesrestriction enzyme sites Linear DNA mapsLinear DNA maps Circular DNA maps (plasmids)Circular DNA maps (plasmids) Maps of inserts within vectorsMaps of inserts within vectors

ApproachApproach

1.1. Determine whether the DNA has Determine whether the DNA has digesteddigested

2.2. Is the digestion complete or Is the digestion complete or partial?partial?

3.3. How many cuts?How many cuts?

4.4. Determine the relative positionsDetermine the relative positions

1.1. Is the DNA digested?Is the DNA digested?

Compare to the Compare to the undigested controlundigested control Which samples were Which samples were

not digested?not digested? 1 and 41 and 4

Which samples were Which samples were digested?digested?

2 and 32 and 3

Ladder

Control

1 2 3 4

2.2. Is the digestion complete?Is the digestion complete?

Complete digestionComplete digestion All the DNA molecules are cleaved at all All the DNA molecules are cleaved at all

the possible sitesthe possible sites Partial digestionPartial digestion

A fraction of the molecules are not A fraction of the molecules are not digesteddigested

Partial undigestedPartial undigested A fraction of the molecules were digested, A fraction of the molecules were digested,

but not at all the possible sitesbut not at all the possible sites Partial digestionPartial digestion

Complete digestionComplete digestion

Digestion

Partial digestion: Partial undigestedPartial digestion: Partial undigested

DigestionNon digested

Partial digestionPartial digestion

Digestionpartial

partial

Is the digestion complete Is the digestion complete or partial?or partial?

Compare to Compare to controlcontrol

Verify the Verify the intensity of the intensity of the bandsbands

Verify the sizesVerify the sizes

Ladder

Control

1 2 3 4

3.3. How many cuts?How many cuts?

Number of sitesNumber of sites Circular DNA = number of bandsCircular DNA = number of bands Linear DNA = Number of bands – 1Linear DNA = Number of bands – 1

4.4. Determine the relative Determine the relative positionspositions

The fragment sizes represent the distances The fragment sizes represent the distances between the sitesbetween the sites

Linear DNA mapsLinear DNA maps

Enzyme Fragments (Kb)

HindIII 3 and 4

SalI 2 and 5

HindIII + SalI 2 and 3

3.0 4.0 HindIII

7.0

HindIII + SalI2.0 2.03.0

Circular DNA maps (plasmids)Circular DNA maps (plasmids)

Enzyme Fragments (Kb)

BamHI 2, 3 and 5

HindIII 1 and 9

BamHI + HindIII 1, 1.5, 2, 2.5 and 3

10.0

7.0

10.0

1.0

9.0 3.0 2.0

1.0

1.5

2.5

Insertion mapsInsertion maps

Recombinant plasmid

Insertion site

Vector

MCS

MCS

ApproachApproach

1.1. Determine the total sizeDetermine the total size

2.2. Determine size of the insertDetermine size of the insert Total size – size of vectorTotal size – size of vector

3.3. Determine the insertion site within the Determine the insertion site within the MCSMCS

4.4. Determine which enzymes cut wihin Determine which enzymes cut wihin the insertthe insert

5.5. Relative mapping in relation to the sites Relative mapping in relation to the sites at known positionsat known positions


Enzyme Fragments

BamHI 7.7Kb

EcoRI 1.0, 3.0, 3.7Kb

PstI 2.0 and 5.7

XbaI 2.7 and 5.0

1.1. Total sizeTotal size• 7.7Kb7.7Kb

2.2. Insert sizeInsert size• 7.7 – 2.7 = 5.0Kb7.7 – 2.7 = 5.0Kb

3.3. Insertion siteInsertion site• Generates 2 Generates 2

fragments of which fragments of which one is the size of the one is the size of the vectorvector

• XbaIXbaI


Enzyme

FragmentsTotal cuts

Sites in vector

Sites in insert

BamHI 7.7Kb 1 1 0

EcoRI 1.0, 3.0, 3.7Kb 3 1 2

PstI 2.0 and 5.7 2 1 1

XbaI 2.7 and 5.0 2 Insertion site

0

Sites to map

Map of PstI : 2 and 5.7KbMap of PstI : 2 and 5.7Kb

5.0

5.7 Kb2.0 Kb

Map of EcoRI: 1, 3 and 3.7KbMap of EcoRI: 1, 3 and 3.7Kb

1.0 3.0

3.7 1.03.0 1.0

P

Molecular Biology Working with DNA. Topics Genomic vs. Vector DNA Purifying plasmid DNA Restriction enzymes Restriction maps.

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