Ch. 9: Presentation Slides

roachavocadoBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

207 views

1

Chapter 4: recombinant DNA

Restriction enzyme analysis

Cloning in
E. coli

plasmids

Transformation

Biomedical application

2

Restriction enzymes


Restriction enzymes cut

double
-
strand
DNA at specific
recognition sequences

which are often 4
-
6 base pair palindromes
= 5’
-
3’ sequence is identical on both DNA
strands


Many restriction enzymes cut the two DNA
strands at different points which
generates complementary single
-
strand
ends =
sticky ends

(others =

blunt ends)

3


BamHI

(from
B. amyloliquefaciens
) recognizes
GGATCC and cuts between the G’s on both strands

Restriction enzymes

4


Restriction enzymes

5

Restriction
enzymes

cut DNA
into defined
pieces,
named
restriction
fragments

Restriction enzymes

6

DNA fragments of different size (e.g.
restriction fragments) can be separated
according to their size by
gel electrophoresis:



agarose

gel electrophoresis (300 bp
-

15 kb)



polyacrylamide

gel electrophoresis (1
-
500 b)

=PAGE

Restriction enzymes

7


Gel electrophoresis

Molecular weigth marker
(band sizes
known)

to compare sample band sizes

8

Recombinant DNA


Sticky ends formed


by restriction enzymes


permit
circularization

or


combinations

of DNA


restriction fragment(s)


by complementary



base pairing



9


A new
combination

of DNA can be made
by combining restriction fragments


Complementary sticky ends can be
covalently linked with
DNA ligase

to form
recombinant DNA


Blunt end DNA fragments (for example
generated by PCR) can also be ligated (but
less efficiently)



Recombinant DNA

10




Ligation of

vector and insert

DNA ligase

Recombinant DNA

11







A
vector

is a replicating unit that can be
opened to insert another DNA fragment



Often
plasmids

are used as vector in
bacteria

A plasmid is a small
self
-
replicating circular

DNA molecule found in bacteria

Recombinant DNA

12






Plasmid vectors have



an
origin

of replication



a
selectable marker

gene (often an antibiotic
resistance)



a
cloning site

or multicloning site (MCS)

Recombinant DNA

13


Transformation by heat shock or
electroshock









bacterium

transformation

Plasmid replication

Replicating
bacteria
form colony


Recombinant DNA

14

Selection




Plate bacteria on selective medium

Select for presence of marker

Medium containing antibiotic

R

Recombinant DNA

15







+

=

Vector

DNA
fragments

Recombinant DNA
molecules

+

=

Recombinant DNA

DNA 1

DNA 2

DNA 3

In reality only one or up
to millions of fragments

16

Cloning = purification






Transform plasmids into bacteria: a cell will replicate
only one plasmid type

Plate bacteria to form colonies

Recombinant DNA

17

Methods of genetic manipulation are named:


Recombinant DNA technology


Genetic engineering


Gene cloning or gene technology

Applications include:


Isolation of specific genes


Production of specific proteins

Genetic engineering


GMO = genetically modified organism,
GMM
= genetically modified

micro
-
organisme




Genetic modification

= targeted
modification of a genetic characteristic
of an organism





transgenic

organisme


Genetic engineering

19

Biomedical applications


Recombinant DNA technology is used to
produce large amounts of medically
important
proteins

such as blood clotting
factors, insulin,…. In either bacteria, fungi,
animal cells, whole animals or plants



DNA probes detect mutant genes in
hereditary diseases

(DNA diagnostics)

20





A
chimeric gene

is constructed of parts of
different genes


An eukaryotic gene can only be expressed
in bacteria when provided with the correct
expression signals

(and vice versa)


Example: human insulin production in
bacteria

Bacterial promoter

Coding region human
insulin gene

Bacterial
terminator

Genetic engineering


Diabetics lack the
hormone insulin


Initially, insulin was
extracted from the
pancreas of cows or
pigs (different protein)


Biotech insulin: safe
and easy

Biomedical applications