Recombinant DNA Isolation / Digestion / Fractionation

Purification of the target fragment Cloning into vectors / Ligation

Transformation of host cell / Selection Replication / Analysis Expression of DNA

Recombination Specifically cut and join DNA

Cut: digestion Join: ligation First steps for cloning

Recombinant DNA

Non Specific DNA breakage

Mechanical shearing

Chemical

- Non specific enzyme

Exonuclease

Endonuclease

Restriction Endonuclease

Mg2+ , ATP and SAM as cofactors Specific recognition site on dsDNA

- Move along 1 5 kb Cut randomly on 1 strand

Require 2nd enzyme to cut another strand

Type I

Restriction Endonuclease

Intermediate properties between types I and IIMg2+ and ATP as cofactors

Specific recognition site Cut both strands

- 2 4 2 6 bp from a recognition site

Type III

Restriction Endonuclease

Mg2+

Specific recognition site Specific cleavage site

within or near recognition site Cut both strands

Fragments with defined length & sequence

Type II

Nomenclature

3 Letters (italic) host genus in upper case host species in lower case

strain or type (non italic) restriction or modification system (Roman numeral)

Eco - RI from E scherichia coli Bam - HI from B acillus amyloliquefaciens Hind - III from H aemophilus influenzae Pst - I from P rovidencia stuartii Sau - 3AI from S taphylococcus aureus Ava - I from A nabaena variabilis

Nomenclature

Target Site of REII

- 48 bases Frequency of the cut

- 4 base cutter-bbbb bbbbbb8

Rotational symmetrypalindrome

Palindrome

Target Site of REII

Restriction Enzyme Break phosphodiester bond

Produce 5’P and 3’OH ends Enzyme: homodimer

1 subunit cut 1 strand away from the axis : overhang / sticky / protruding

at the axis : blunt end

5 ’ P and 3 ’ OH ends

5’ sticky end

3’ sticky end

blunt end

Source microorganism Enzyme Rec. Site Ends

Arthrobacter luteus Alu I AGCT Blunt Bacillus amyloiquefaciens H Bam HI GGATCC Sticky

Escherichia coli Eco RI GAATTC Sticky Haemophilus gallinarum Hga I GACGC(N)

5 Sticky

Haemophilus infulenzae Hind III AAGCTT Sticky Providencia stuartii 164 Pst I CTGCAG Sticky

Nocardia otitiscaviaruns Not I GCGGCCGC Sticky Staphylococcus aureus 3 A Sau 3 GATC Sticky

Serratia marcesans Sma I CCCGGG Blunt Thermus aquaticus Taq I TCGA Sticky

Restriction Enzyme

- Sequence specific tails DNA with compatible ends: join

Different sources Different enzymes

Restriction Enzyme

Different enzymes : Different recognition sites Produce compatible ends: Get rDNA

Sau3AI 5 ’ NNN”GATC 3NNN ’BamHI 5’ NNNG”GATCC 3NNN ’

Recombinant DNASau3AI 5’ NNN GATC 3NNN ’ 3’ NNNCTAG 5NNN ’BamHI 5’ NNNG GATCC 3NNN ’ 3’ NNNCCTAG G 5NNN ’rRNA 5’ NNN GATCC 3NNN ’ 3’ NNNCTAG G 5NNN ’

5’ NNNGATCC 3NNN ’ 3’ NNNCTAGG 5NNN ’ recut by Sau 3AI / Bam HI ?

Restriction Enzyme

Methylation Modification of recognition site

Effect on cuttingBamHI * GGATC5mC * 6mATCC

**GGATC4mC **GGAT5CC

Restriction Enzyme

Isoschizomer

Different host same recognition site same or different cleavage site

Restriction EnzymeIsoschizomerXho I / PaeR7I

5’ NNNC”TCGAG 3NNN ’SmaI 5’ NNNCCC”GGG 3NNN ’XmaI 5’ NNNC”CCGGG 3NNN ’

Restriction Enzyme

Isoschizomer: methylation sensitivity

Hpa I (X ) and Msp I (/)C5Cbb

Sma I (X ) and Xma I (/)CC5Cbbb

Restriction Enzyme

Basic tool for creating rDNA Restriction Map

Applications

Restriction Map

Locations of recognition sites on DNA Highly specific fragments

- Digestion with 6 8 base cutter Size fractionation on gel

Different patterns from different enzymes Single and Double digests

Restriction Map

Restriction Map

Restriction Map

Ligation

E. coli ligase NAD+ as cofactor Sticky ends

To ligate by Ligase

- T4 infected E. coli ligase ATP as cofactor

Blunt or sticky ends

Ligation

Seal nicks on dsDNA Form phosphodiester bond

Require 5’ P and 3’ OH

Ligation

Ligation

Example???

Ligation

Intramolecular ligation(re)circularization

Ligation

For recombinant DNA increase DNA concentration increase temperature (Ti)

dephosphorylation

Dephosphorylation

Other Enzymes

DNA polymerase RNA / DNA dependent DNA polymerase

Other Enzymes

E. coli DNA polymerase DNA dependent DNA polymerase

-- 5 3’ > ’ polymerase-- 5 3’ > ’ exonuclease-- 3 5’ > ’ exonuclease

E. coli DNA Polymerase

E. coli DNA Polymerase

DNA polymerase: Klenow fragment-- 5 3’ > ’ polymerase -- no 5’ >3’ exonuclease-- 3 5’ > ’ exonuclease Active on ds DNA

Klenow Fragment

T4 polymerase

DNA polymerase: T4 polymerase-- 5 3’ > ’ polymerase -- no 5’ >3’ exonuclease-- 3 5’ > ’ exonuclease Active on ss DNA

Taq polymerase

DNA polymerase: Taq polymerasethermostable

- 7 5 8 0 C PCR reaction

Reverse transcriptase

RNA dependent DNA polymerase

Exonuclease III

Kinase

Phosphatase

Vector

b bbbbbb bbbbbbb Carry / Multiply specific DNA fragments

Cloning sitemultiple cloning region / polylinker

Origin of replication (ori)Selectable marker

Plasmid vector

Bacterial minichromosome (2-20 kb)not linked to main chromosome

Autonomous replication (replicon)Mostly ds circular formSome with linear form (actinomycete / spirochete)Take up 100 bp – 10 kb

Plasmid vector

Carry antibiotic-resistance genesproviding selectable phenotype to host

Other selectable markerssugar fermentationheavy metal resistancehydrogen sulfide productionenterotoxin production

Plasmid vector

Insertional inactivationwhen MCR in selectable-marker gene

High copy number plasmidreplicate 10-200 copies per bacterial cycle

Low copy number plasmidone or a few copies

Plasmid vector

Ideal propertiesLow molecular weight

easy to handle, separate & purifyHigh copy numberUnique restriction sitesDisabled: no survival outside lab

Plasmid vector

Plasmid cloning

Bacteriophage vector

Bacterial virus: forming plaqueLinear or Circular shapeCohesive ends (cos, short ss 5’ protruding)Lytic / Lysogenic cycle

Phage cycle

Bacteriophage vector

Central section of phage DNAFor integration into host chromosomeNot necessary for replicationReplaced by foreign insert

Left and Right arms: easily isolatedEssential for replication and packaging

Bacteriophage vector

Bacteriophage vector

Require certain size for maturationand packaging

Engineered to be safe andhave MCR

Available for big DNA insert

Phage packaging

Phage cloning

Plaque lift / Hybridization

Cosmid

Plasmid / Phage hybridplasmid: ori and selectable markerphage: cos site for packaging

For large insert (32-47 kb)

Cosmid

Cosmid cloning

Cosmid cloning

Yeast Artificial Chromosome

Big inserts: hundreds of kbCloning elements

Yeast sequencesSelectable markersMCR

Yeast Artificial Chromosome

YAC cloning

Expression vector

Available for screening of gene productof cDNA insert

MCR within transcription regionsbetween promoter and terminator

Bidirectional cloning for correct orientationForeign or Fusion proteins