Summary Genome technology and applications

H1 : CELL BASED DNA CLONING
1.1 PRINCIPLES OF DNA CLONING

1.1.1 CELL BASED DNA CLONING

4 steps of cell based DNA cloning :

1) In vitro construction of a recombinant DNA molecule
2) Transformation in a host
3) Selective propagation of clones
4) Isolation of recombinant DNA clones




STEP 1 : IN VITRO CONSTRUCTION OF A RECOMBINANT DNA MOLECULE

Recombinant DNA molecule = combining different DNA molecules

→ requires cutting and pasting of DNA

● Cutting = restriction endonucleases
● Pasting = DNA ligase

Requires a replicon

● A piece of DNA that makes independent DNA replication possible
● Region of an organism’s genome that independently replicates from a single origin of replication (ORI)
● Replicon is specific for a host

Plasmid

● Mostly we clone in bacteria → replicon = plasmid
● Plasmid is a small circular DNA molecule found in bacteria
● They are physically separated from chromosomal DNA and replicate independently

Vector

● Usually a construct called “vector” is used, containing many features used in the cloning process
● Vector = plasmid that has been engineered → special features have been added
● It’s used as a vehicle to carry a particular DNA segment into a host cell



Schema

1) Bacterial cells with vector = plasmid
● Purify plasmid
● Cut it with restriction enzyme
2) Human cells
● Purify DNA
● Cut it with restriction enzyme
3) Ligate bacterial DNA with human DNA = recombinant DNA

,STEP 2 : TRANSFORMATION

Transformation = recombinant DNA molecule is introduced in a host cell

● Usually introduction in bacterial cells or yeast
○ Easy to grow
○ Fast reproduction
● For expression studies, cloning is often done in eukaryotic cells
○ Mammalian cells, insect cells → see later in this chapter
● So take construct → put it in host cells = transformation




STEP 3 : SELECTIVE PROPAGATION OF CLONES

● The transformed cells are plated on agar → each individual cell forms a colony
● 1 colony can be grown in liquid medium to obtain more cells
● Each colony consists of clones
○ All cells are identical because they have the same ancestor cell




STEP 4 : ISOLATION OF RECOMBINANT DNA CLONES

Cell lysis and purification

● 1 colony is picked up from the agar and will be grown in liquid medium → grow it overnight → liquid
medium with a lot of bacteria → lyse them → purify recombinant DNA




→ now look back for more details

,1.1.2 RESTRICTION ENDONUCLEASES

→ First step : making recombinant DNA molecule

Restriction endonucleases (RE) = enzymes used for cutting to make recombinant DNA

● Nomenclature: 1 letter genus, 2 letters species, followed by number
○ E.g. HaeIII: Hemophilus aegypticus
○ The RE is named after a bacteria because we get these enzymes from bacteria
○

Function of RE in bacteria = defense mechanism against bacteriophages

● Bacteria can get infected by viruses = bacteriophages
● DNA from bacteriophage enters the bacterium and is cleaved
● Type II RE will cut a specific recognition sequence
● RE recognises a specific DNA sequence BUT we don’t want the bacterial DNA to be cut
○ There’s a matching sequence specific DNA methylase
○ It methylases the recognition sequence in the bacterial DNA → bacterial DNA cannot be cut

Recognition sequence

● Usually 4-8 bp
● Usually palindrome



Cleavage

● On the symmetry axis: blunt ends
● Not on the symmetry axis : overhangs = sticky ends = cohesive termini
● Sticky ends can base pair and form unstable double helices by DNA ligase
○ 5’ overhang
○ 3’ overhang

, Examples of restriction enzymes that are used in the lab

MBO1 and BAMH1 create the same overhangs

● 5 prime overhang = GATC
● These overhangs are compatible = They can be ligated




Examples of RE and the results they give on total human DNA

➔ Take the whole human genome and cut it with an RE = you get fragments of a certain length (bp)
➔ If you have a short recognition sequence you get short fragments



1) HaeIII gives longer fragments than AluI, even though the recognition sequence is equal in length (4 bp)
● The human genome has less GC than AT
● There will be less sites for HAEIII than for AluI
● Therefore the fragments of AluI will be shorter than HAEIII fragments



2) TaqI gives even longer fragments
● It has the same GC content than AluI but gives longer fragments
● Has to do with CpG → important site for DNA methylation
● CpG are only ⅕ of the frequence of a dinucleotide = rare in the human genome




Isoschizomers: different restriction enzymes that have the same recognition sequence

Some RE have compatible cohesive termini → E.g. BamHI: GGATCC en MboI: GATC