BIL 333 Lecture I Plasmids v Small, circular, double-stranded DNA v Exogenous to genome! v Origin of Replication v Marker Gene v ( Reporter Gene ) Plasmids v Marker Gene Changes Phenotype Of Host v (Antibiotic Resistance) v Used for screening for successful introduction into cell.. Useful Plasmids v Introduced Into Host Cells - Transformation Useful Plasmids v Genes Inserted at Restriction Site Useful Plasmids v ( Transfection ) v Plasmid = Vector v Cell must first be rendered competent v Insert v Recombinant Construct 1
Restriction Endonucleases Nucleases v Enzymes that cut DNA and RNA Exonucleases-Cut linear fragments at the ends Endonucleases- cut either linear or circular nucleic acids at sites internal to the molecule Restriction Endonuclease v Endonucleases whose function depends on a specific DNA sequence. Haemophilus influenzae = HindII 5 GT(pyrimidine)(purine)AC 3 3 CA(purine)(pyrimidine)TG 3 v Restriction Enzymes Type I : nonspecific cleavage Type II: palindromic sequences*** Type III: nonpalendromic sequences Restriction Sequences 2
Type II Restriction Endonucleases Can cleave to leave overhanging ends or blunt ends ( Sticky End Cutters, or Blunt End Cutters ) Sticky vs. Blunt Ends Palindrome v Inverted Sequence Repeat v (Both strands have same sequence if read in same polarity) Palindrome Palindrome v Madam I m Adam v 5--AACGGCCGTT--3 v 3--TTGCCGGCAA--5 5--AACGGCCGTT--3 3--TTGCCGGCAA--5 3
Palindromes can form Cruciforms Palindromes can form Cruciforms Type II Restriction Endonucleases Type II Restriction Endonucleases v Different DNA strands cut with the same Restriction Enzyme.. v Generate the same sticky ends.! Recombinant DNA Isoschizomers v Different Endonucleases that recognize the same sequences and cleave at the same position. EcoRI 5 G/AATTC 3 FunII 5 G/AATTC 3 4
Neoschizomers v Different Endonucleases that recognize the same sequence but cleave at different positions within that sequence. Sma 1 5 CCC/GGG 3 Xma 1 5 C/CCGGG 3 Methylase and Nuclease v Late 1960 s, Stewart Linn and Werner Arber identified enzymes in bacteria responsible for phage growth restriction One enzyme cleaved DNA at a wide variety of locations along the length of the molecule (restriction endonuclease) One enzyme methylated DNA (modification methylase) Methyl group protrudes into major groove of DNA, blocking the restriction enzyme s binding site. Modification & Restriction Methylation (Modification) & Restriction v Typically - pairs of Restriction/ Modification enzymes recognize the same restriction sequence.. v Impart protection from cleavage ( restriction ) Source of Methyl - SAM SAM 5
Restriction Endonuclease Reactions v Enzymes are sold stabilized in glycerol Keep glycerol less than 5% of total volume in all restriction reactions Restriction Endonuclease Reactions v Enzyme unit Enzyme required to completely cut 1µg of λ DNA in a 50 µl reaction at 37 o C for 1 hour. v Need compatible reaction buffer that contains coenzyme DNA Agarose Gel Electrophoresis Migration of DNA in Agarose v DNA usually cut into fragments using restriction endonucleases prior to electrophoresis v (+) Electrode on Bottom Of Gel! (DNA phosphates are (-)) v Mobility is inversely proportional to Log 10 of MW Log 10 of MW Distance Migrated (mm) Shape Also Effects Mobility Supercoiled Plasmids v More Bulky Shapes v Shape Matters v More Friction v Slower Mobility v (M α MW / friction) Open Circle ( Nicked ) SC Biologically isolated plasmids usually mixture of SC and OC 6
Supercoiled Plasmids Topoisomers.. Double Strand DNA Supercoils Gyrase Open Circle Uncut (unrestricted) DNA Migrates Differently SC OC Linear v Circular forms (plasmids) of DNA migrate distinctly differently than linear DNA of the same size Most preps of uncut plasmid contain at least two different forms Other factors affecting mobility SS Nick Open Circle v Agarose Concentration v Voltage Large fragments migrate proportionally faster under higher voltage v Electrophoresis Buffer TAE (Tris-acetate-EDTA) TBE (Tris-borate-EDTA) v Ethidium Bromide Alters mass and rigidity DS Nick Linear 7
Ethidium Bromide Intercalation Fluorescence v Chromophores can absorb hv v After Absorbance? Energy released as heat v Chromophore - Abs hv v Fluorophore - Releases absorbed hv energy as florescent light Energy released as light ( fluorescence ) 8
v EtBr : v Releases abs light (fluorophore) v Does not absorb much UV light (not chromophore) v DNA: v Absorbs UV light (chromophore) v Does not release fluorescense (not a fluorophore) v DNA Absorbes UV EtBr Intercalation v Transfers UV energy to intercalated EtBr ( FRET ) v Energy released from EtBr as fluoresence Images DNA! λ Phage v Bacteriophage with double-stranded DNA chromosome of 48,502bp λ DNA v Can exist as a circular molecule as well as a linear molecule 48,502 BP 48.5 KB Lambda HindIII Restriction Map 9
Lambda HindIII Restriction Map Gels Gone Bad.! Gels Gone Bad.! 10
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