Page 30 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

324 Chapter 9 Digital Analysis of DNA

(a) EcoRI site Figure 9.4 Creating recombinant DNA molecules with
plasmid vectors. (a) A plasmid vector contains three main
features: (i) an origin of replication; (ii) a selectable marker gene
conferring resistance to an antibiotic like ampicillin (orange); and
A G G A A T T C C C (iii) a synthetic polylinker (purple) containing restriction enzyme
T C C T T A A G G G sites unique to the plasmid, such as EcoRI. (b) EcoRI enzyme cuts
the plasmid vector at its single EcoRI recognition site, opening up
the plasmid circle. EcoRI cuts human genomic DNA at many sites,
Polylinker producing a mixture of fragments, only one of which is shown
amp r (green). The vector and the human genomic fragments all have the
Vector same EcoRI sticky ends. (c) The enzyme-cut plasmid and human
genomic DNA fragments are mixed together in the presence of
the enzyme DNA ligase, which sutures them to each other to form
circular recombinant DNA molecules. Note that the recombinant
Origin of DNA now has two sites for EcoRI.
replication
Digest EcoRI fragment
with EcoRI enzyme of human DNA
5'
(b) AA TT C AA G C G 3'
5'
3' A A T 3' G T T C G C T T A A
G A A T C 5'
G T T 5' C
A C C 3' G G C Sticky Sticky
T G end end
Sticky ends

DNA
ligase

(c) EcoRI site

A A T T C A A
G A A G T T G
G T T C C G
A C C G A
T C T A EcoRI site
T T
A T
Human DNA insert A G C C

G
Recombinant DNA G C
molecule