Essentials of molecular cloning

pumpkincentersulkyBiotechnologie

16 déc. 2012 (il y a 4 années et 9 mois)

117 vue(s)

인간의

삶과

역사

속의

미생물

2010
-
2
학기

강의자료

ppt
-
8

미생물


어떤

존재인가
?

유전공학
(genetic engineering)


가능하게



미생물

The use

of
in vivo
techniques in the isolation,
manipulation, alteration, and expression of DNA
(or RNA) and in the development of genetically
modified organism

* Recombinant DNA technology
(
재조합

DNA
기술
)


Biotechnology


use of organisms to form useful products


Genetic engineering (
유전공학
)



Basic
techniques
include:


-

Restriction enzymes (restriction
endonuclease
,
제한


효소
)


-

Cloning vectors (
클로닝매개체
)


-

Molecular cloning (
분자클로닝
)


Restriction

and modification enzymes



Restriction enzymes
:


Protect prokaryotes from hostile foreign DNA (e.g.,
viral genomes)


Widespread among prokaryotes


Rare in eukaryotes


Recognize specific DNA sequences and cut DNA at
those sites


Essential
for
in vitro

DNA manipulation


Restriction

and modification enzymes

Restriction

and modification of DNA



Modification enzymes
: protect cell’s DNA for
restriction enzymes


Chemically modify
nucleotides in restriction
recognition sequence


Modification generally consists of
methylation

(addition

of methyl group) of
DNA

Restriction

and modification of DNA


Molecular
cloning
: isolation and incorporation of
a piece of DNA into a
vector

so it can be
replicated and manipulated


Three main steps of gene cloning

1)
Isolation and fragmentation of source DNA

2)
Inserting DNA fragment into cloning vector

3)
Introduction of cloned DNA into host organism



Essentials of molecular cloning

1) Isolation and fragmentation of source DNA


Source DNA can be genomic DNA, RNA, or PCR
amplified fragments


Genomic DNA must first be restriction digested


Essentials of molecular cloning

2) Inserting DNA fragment into cloning vector


Most vectors are derived from
plasmids

or
viruses


DNA is generally inserted
in vitro


DNA
ligase
: enzyme that joins two DNA molecules


Works with sticky or blunt ends


Essentials of molecular cloning

3) Introduction of cloned DNA into host organism


Transformation is often used to get recombinant DNA
into host


Some cells will contain desired cloned gene, while
other cells will have other cloned genes


Gene library
:

mixture of cells containing a variety of
genes


(= genomic library)


Shotgun cloning
: gene libraries made by cloning
random genome fragments


Essentials of molecular cloning

Essentials of molecular cloning


Plasmids are natural vectors and have useful
properties as cloning vectors


Vector
transfer carried out by chemical
transformation or
electroporation

(
전기천공
)

Plasmids as cloning vectors

Hosts for molecular cloning (
microorganisms
)

Steps for cloning a gene

Restriction enzymes and their recognition sequences

Recombinant plasmid construction

Genomic library construction and screening by
phenotyphic

rescue


In
medicine


many useful proteins


gene therapy


In
agriculture


use of genetic engineers allows for the direct transfer
of desirable traits to agriculturally important animals
and plants


Applications of genetic engineering


There
is scientific and philosophical concern about


the use of
embryonic stem cells

in gene therapy. This
is currently a major controversial
issue


the production of genetically engineered food


the possibility of the production of genetically
engineered organisms by bioterrorists


other issues



Social impact of genetic engineering