Summary Cloning introduction - cell-based DNA cloning

GENOME TECHNOLOGY AND
APPLICATIONS: CLONING
INTRODUCTION: CELL-BASED
DNA CLONING
PRINCIPLES OF DNA CLONING

DNA cloning can be cell based: it happens in 4 steps

1. In vitro construction of a recombinant DNA molecule
a. Recombinant = combining different pieces of DNA, bv animal and human
2. Transformation
a. Bacteria can take up foreign DNA
b. It is a key step in DNA cloning
c. Occurs after restriction digest and ligation and transfers newly made plasmids into bacteria
3. Selective propagation of clones
a. After transformation, bacteria are selected on antibiotic plates
b. Bacteria with the plasmid are antibiotic-resistant  will form a colony
4. Isolation of recombinant DNA clones

Colonies with the right plasmid can be grown to make large cultures of identical bacteria = used to produce
plasmid or make protein

DNA cloning = The process of making multiple, identical copies of a specific piece of DNA. The gene or other
DNA fragment of interest is first inserted into a circular piece of DNA = a plasmid. The insertion is done by ‘cut
and paste’ enzymes. The produce a molecule of recombinant DNA, or DNA assembled out of fragments from
multiple sources.

The recombinant plasmid is introduced into bacteria. The bacteria
that carry the plasmid are selected and grown up (they are AB-
resistant). As they reproduce, they replicate the plasmid and pass
it on to their offspring, making copies of the DNA it contains
(more plasmid = more DNA/gene of interest).

Used for: Bv the human insulin gene is expressed in E. Coli
bacteria to make insulin used by diabetics. The plasmid-carrying bacteria are used as ‘factories’ to make
protein.

The 4 steps of DNA cloning
1. In vitro construction of a recombinant DNA molecule

Requires: cutting + pasting of DNA

 Restriction endonucleases (restriction enzymes)
 DNA ligase

,Requires: a replicon

 A piece of DNA that makes independent DNA replication possible
 Replicon : host-specific
 Replicon : a construct called vector = containing many features used in the cloning process




2. Transformation

Transformation = Recombinant DNA molecule is introduced in a host cell

 Host cell = mostly yeast or bacterium
o Easy to grow
o Fast reproduction
 For expression studies  cloning is often done in eukaryotic cells (mammalian/insect cells)
o For expression studies, cloning in a bacterium usually precedes (het gaat vooraf aan) cloning
in the host used for the expression
o So they also need cloning in bacteria, cloning in bacteria is needed (almost) every time: the
cloning in bacteria is a preparation for cloning in eukaryotic cells (when everything is ready,
the clones are put in the eukaryotic cells)

, 3. Selective propagation of clones

Cells are plated on agar

- Each individual cell forms a colony
- Each colony is a clone: all cells are identical + have the same ancestor cell
- 1 colony can be grown in liquid medium  to obtain more cells

If the colonies are to small (left plate), you cannot identify the
colonies




4. Isolation of recombinant DNA clones

Now the cells have formed colonies on the agar plate

The recombinant DNA is purified from the cells

Last step: lysis and purification

- U take out the construct, you can do this with a sterile pen
- Purify the recombinant DNA clones
- U take the recombinant DNA clones out of the identical cell clones