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A fragment of DNA that has been introduced into a foreign cell

This results in exact copies of the original DNA fragment


being made when the foreign cell replicates and divides
Gene cloning became possible after Watson, Crick and other
researchers began to discover the way molecules transmit
genetic information.
In the 1970s, this technique
was finally able to be carried out.
Genetic structures which have the ability to replicate
independently of the chromosomes (typically a small circular DNA strand in the
cytoplasm of a bacterium or protozoan)

They possess a copy number- # of copies of particular plasmid


found in a bacterial cell

1-chromosomes
2-plasmids
Enzymes which slice DNA into fragments at specific
sequences (4-8 base pairs long)
When sliced at the ends, the DNA becomes sticky if there is
a single-strand exposed (overhang)
Certain R E need to be used to ensure the gene being analyzed
is removed completely from the DNA
Multiple R E are being used at the same time
Also called restriction enzymes

1962: molecular scissors discovered in in bacteria

E. coli bacteria have an enzymatic immune system that


recognizes and destroys foreign DNA

3,000 enzymes have been identified, around 200 have


unique properties, many are purified and available
commercially
Named for bacterial genus, species, strain, and
type

Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
Enzymes recognize specific 4-8 bp sequences

Some enzymes cut in a staggered fashion - sticky ends

EcoRI 5GAATTC3
3CTTAAG5

Some enzymes cut in a direct fashion blunt ends

PvuII 5CAGCTG3
3GTCGAC5
Restriction Enzymes for Transformation
Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI
5-C-G-G-T-A-C-T-A-G-OH PO4-A-A-T-T-C-A-G-C-T-A-C-G-3
3-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4 + HO-G-T-C-G-A-T-G-C-5

Complementary base pairing

5-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5

+ DNA Ligase, + rATP

5-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5

recombinant DNA molecule


Recombinant DNA is
constructed using restriction
enzymes
Important: In order to join 2
pieces of DNA together they
have to be cut by the same
restriction enzyme
Why?
Otherwise, the sticky ends wont
match DNA cant bind together