15 - Molecular Cloning Flashcards
Uses of DNA cloning
- Gene mapping
- DNA sequencing
- Identification of unknown genes
- Allows manipulation of DNA
Process of cloning gene
- Choose strategy for isolating gene of interest
- Choose a destination (e.g. plasmid)
- Choose destination host (e.g E. coli)
- Choose method for identifying recombinant E. coli carrying gene of interest
Two basic strategies for isolating gene from genomic DNA
- Cloning random restricted DNA fragments (library of randomly cloned fragment)
- Cloning specific PCR fragment (single cloned fragment)
Advantages of cloning randomly restricted fragments
Do not need to know the sequence of the target gene
Disadvantages of cloning randomly restricted fragments
- Not specific for gene of interest
- RE may cut inside gene of interest
Advantages of cloning specific PCR fragment
Very specific amplification of gene
Disadvantages of cloning specific PCR fragment
Must know sequence of target gene for design of primers
Tools required for cloning random DNA fragments
- High molecular weight DNA
- Restriction enzymes
- T4 DNA ligase
- Bacterial transformation
Restriction enzymes
Enzymes that can cleave the
phosphodiester bond at specific sites in the DNA duplex
Properties of restriction enzymes
- Named according to source
- Require Mg++ for activity
- Cut at fixed position within restriction site (sticky or blunt ends)
Restriction site
4-10 base pair palindrome where restriction enzymes cut
Frequency of cutting of restriction enzymes
Related to the length of the recognition sequence
Why is buffer of pH 8 important
To ensure DNA remains negatively charged
Which node does DNA migrate to in electrophoresis
Anode (positive)
What do plasmid cloning vectors contain
- Selectable marker
- Multiple cloning site
- Origin of replication
Mechanism for detecting recombinant vectors
- Insertional inactivation (blue white screening)
- Gain of function (phenotype e.g antibiotic resistance)
Ligation
Creation of a phosphodiester bond between the 3’ hydroxyl of one nucleotide and the 5’ phosphate of another
Requirements for ligation
- DNA compatible ends
- T4 DNA ligase from T4 phage
- ATP
- Mg++
- Optimal buffer pH
- Incubation at 16ºC
Mole ratio of vector to insert for sticky ligation
1:3
Mole ratio of vector to insert for blunt ligation
1:1
Compatible ends
- All blunt ends
- Sticky ends that are identical
How can sticky ends be made into blunt ends
Fill in 5’ overhangs or removal of 3’ overhangs by klenow fragment or T4 DNA polymerase
Halting of self ligation of vector
- Alkaline phosphatase removes 5’ phosphates from termini of cut DNA
- Must be removed before ligation
Sources of alkaline phosphatase
- Bacterial
- Calf intestinal
- Shrimp
Expression of cloned gene in new bacterial host
- DNA ligation (formation of clones)
- Transformation into E. coli
- Bacteria plated on medium + antibiotic
- Overnight incubation
- Screen for gene expression
Isolation of plasmid
- Screen phenotype (e.g haemolysis)
- Pick colony
- Grow pure culture
- Purify the plasmid (Alkaline lysis method/column purification)
- Analyse plasmid (restriction map/sequence)
Steps of blue white screening
- Digest foreign DNA and pUC18 with Sall
- Ligate digested DNA together
- Transform into E. coli host
- Plate onto agar plates containing ampicillin
- White colonies contain recombinant vector, blue contain self ligated vector
β-galactosidase LacZ
- Cleaves lactose to galactose and glucose
- Cleaves X-gal which forms blue precipitate
Promotor P-lac
- Binds repressor Lacl
- Transcription is induced by allolactose
Blue white screening
- LacZ can be cleaved into two fragment peptides
- LacZΩ and LacZα are BOTH
ENZYMATICALLY INACTIVE by themselves - When co expressed, the peptides bind to each other to restore β-galactosidase activity
- Both genes are under the control of LacI and transcription is induced using isopropyl-β-thiogalactoside
E. coli host strain encodes only LacZΩ
- In its genome or on a plasmid
- Not resistant to antibiotics unless it gets cloning plasmid
- LacZ is not functional and no colony is formed
Empty vector used for cloning encodes LacZ⍺
- The multiple cloning site is inside the gene encoding LacZ⍺
- When empty vector is transformed into E. coli host strain, complementation occurs
- LacZ is functional and blue colony is formed
Recombinant vector does not produce LacZ⍺
- The insert integrates into the multiple cloning site
- Gene encoding LacZ⍺ is disrupted
- LacZ if not functional, white colony formed
Overall process of cloning
- Obtain vector
- Obtain insert (PCR or fragmentation)
- Restriction digest of vector and insert
- Ligate insert and vector (T4 DNA ligase)
- Transform ligation mixture in E. coli
- Selection (blue white screening)
- Verification (sequencing)
Methods of transformation (vector into host)
- Heat shock
- Electroporation
