Download Chapter 12: Biotechnology 1. Recombinant DNA What is

Chapter 12:
Biotechnology
1. Recombinant DNA
2. Recombinant DNA Applications
1. Recombinant DNA
What is “Recombinant DNA”?
The joining of DNA from different sources.
This can happen in nature (in vivo)…
• the transfer of DNA involving bacteria or viruses
…or in the laboratory (in vitro)
• the cutting & splicing of DNA fragments by
molecular biologists
The term “recombinant DNA” generally refers to
laboratory kind…
1
Recombinant DNA
in Nature
Bacteria can acquire DNA
from an outside source by
a process known as
transformation
• chemical treatment of bacterial
cells followed by a brief heat
treatment can induce the cells
to internalize & retain the DNA
Plasmids
Bacteria are transformed
more commonly by small,
circular DNA fragments
called plasmids
• taken up from outside or
transferred from one
bacterium to another
Restriction enzyme
recognition sequence
1
Recombinant DNA
in the Laboratory
GAATTC
CTTAAG
DNA
Restriction enzyme
cuts the DNA into
fragments
G
CTTAA
2
The cutting and splicing of
DNA in vitro involves the use
of restriction enzymes (RE’s):
A AT TC
G
Sticky end
Addition of a DNA
fragment from
another source
A AT TC
3
G
Two (or more)
fragments stick
together by
base-pairing
4
G A AT T C
C T TA A G
G A AT T C
C T TA A G
DNA ligase
Pastes the strand
5
Recombinant DNA molecule
G
CTTA
e.g.
A
EcoR I cuts at:
..GAATTC..
..CTTAAG..
Hind III cuts at:
..AAGCTT..
..TTCGAA..
There are many different RE’s,
each cutting a different sequence
2
“Cloning” a Gene
Human cell
E.coli
1 Isolate DNA
from two sources
2 Cut both DNAs
with the same
restriction enzyme
DNA
Plasmid
Gene V
DNA “sequence of interest”
is inserted into a plasmid &
“carried” inside bacteria
Sticky ends
3 Mix the DNAs;
they join by
base-pairing
1) cut plasmid and DNA to
be cloned with same RE
2) ligate fragments together
using DNA ligase enzyme
4 Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
5 Put plasmid into bacterium
by transformation
Recombinant
bacterium
3) transform bacteria, select
for “clones” with plasmid
**In this form the “DNA of interest”
can be easily multiplied and purified**
6 Clone the bacterium
Bacterial clone carrying many
copies of the human gene
PCR Amplification
The creation of recombinant
DNA molecules in the
laboratory requires large
amounts of the DNA
fragments to be combined:
• the PCR (Polymerase
Chain Reaction) technique is
routinely used to generate
huge numbers of identical
DNA fragments
• involves in vitro DNA
replication to amplify
desired DNA sequences
exponentially
2. Recombinant DNA
Applications
3
Useful Applications Involving
Recombinant DNA Technology
DNA sequencing, fingerprinting
• involves the separation of DNA fragments by size
using gel electrophoresis
Commercial Production
• using recombinant bacteria, yeast to make “lots” of
a protein (e.g., insulin)
Gene therapy
• replacing defective genes (still experimental)
Transgenic organisms (GMO’s)
• putting “desirable” genes into animals, plants
Gel Electrophoresis
–
Mixture of DNA
molecules of
different sizes
–
Longer
molecules
Power
source
Gel
+
Shorter
molecules
+
Completed gel
DNA fragments of different lengths generated by RE’s or
other methods are separated by size by “running a gel”
• used in DNA sequencing, DNA fingerprinting and
numerous other techniques
Transgenic
Organisms
Potato plant
containing pest
resistance gene
Salmon
containing extra
growth hormone
gene
**Transgenic
organisms contain
a foreign gene**
4
Key Terms for Chapter 12
• recombinant DNA
• transformation
• plasmid
• restriction enzymes
• PCR
• gel electrophoresis
• transgenic
Relevant Review Questions:
2-4, 6
5