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Chapter 14
Genetic Recombination and
Genetic Engineering
The biochemistry and molecular
biology department of CMU
Section 1
DNA Recombination
DNA recombination
• Homologous Recombination
• Conjugation
• Transformation
• Transduction
• Site-specific Recombination
• Transposition
§1.1 homologous Recombination
• Homologous recombination occurs
between identical or nearly identical
sequences. It is also called general
recombination.
5´
3´
3´
5´
3´
5´ endonuclease
5´ (recBCD)
3´
5´
3´
3´
5´
3´
5´
5´
3´ 5´
3´
DNA invading
(recA)
5´
3´
3´
5´
3´
5´
5´
3´
endonuclease
(recBCD)
5´
3´
3´
5´
3´
5´
5´
3´
Branch
migration
(recA)
5´
3´
3´
5´
3´
5´
5´
3´
DNA
ligase
5´
3´
3´
5´
3´
5´
5´
3´
Holiday intermediate
Holliday intermediate
5´
3´
3´
5´
3´
5´
5´
3´
3´
5´
3´
5´
3´
5´
3´
5´
5´
3´
endonuclease
(ruvC)
3´
5´
5´
3´
DNA ligase
5´
3´
5´
3´
5´
3´
3´
5´
5´
3´
3´
5´
3´
5´
splice
recombinant
3´
5´
endonuclease
(ruvC)
3´
5´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
DNA ligase
5´
3´
patch
recombinant
5´
3´
3´
5´
§1.2 Conjugation
• Bacterial Conjugation has been defined
as the transmission of genetic information
from a donor bacterium to a recipient cell
through cell-to-cell contact.
Conjugation process
Conjugation process
Conjugation process
§1.3 Transformation
 Introduction of an exogenous
DNA into a cell, causing the cell to
acquire a new phenotype.
Transformation
DNA
Transformation experiment of
Streptococcus pneumoniae
§1.4 Transduction
• Transduction is the transfer of DNA
fragments from one bacterium to
another bacterium by a
bacteriophage.
Transduction
§1.5 Site-specific Recombination
• Site-specific recombination occurs at a
specific DNA sequence.
• The first example was found in the
integration between l DNA and E. coli
DNA.
λDNA integration
Phase variation of Salmonella
typhimurium flagella
hix
hix
hin
P
P22
H2
rH1
×è¶ô»ùÒò
P1
H1
DNA
H2 flagellin
Hin
repressor
P2
P2
hin
H segment
H2
rH1
×è¶ô»ùÒò
P1
H1
H1 flagellin
Recombination signal sequence (RSS)
CACAGTG (12/23) ACAAAAACC
GTGTCAC
TGTTTTTGG
RSS
Recombination activating gene enzyme
(RAG1 and RAG2)
§1.6 Transposition
• Transposition is the movement of
specific pieces of DNA in the genome.
• Transposition resembles site-specific
recombination being catalyzed by
special enzymes.
IS Transposition
insertion sequences (IS) including:
inverted repeats (IR)：9~41bp
transposase gene
repeated sequences：4~12bp
Transposase gene
types of IS transposition
• duplicative transposition
• Conservative transposition
duplicative transposition
Conservative transposition
transposon
• Insertion sequence + another
gene (usually antibiotic gene)
Transposase gene
tet-R gene
Transposons Transposition
Section 2
Recombinant DNA
Technology
§2.1 Correlative concepts
Clone
A clone is defined as a number of
identical copy (molecules, cells or
individuals) all derived from a common
ancestor. Also named asexual
multiplication.
DNA Cloning
DNA cloning involves separating a
specific gene or segment of DNA from
its larger chromosome and attaching it
to a small molecule of carrier DNA,
then replicating this modified DNA
thousands or even millions of times.
Recombinant DNA technology
• By artificial means, when a gene of
one species is transferred to another
living organism, it is called
recombinant DNA technology. In
common parlance, this is known as
genetic engineering.
Applications in enzymology
• restriction endonucleases
• DNA polymeraseⅠ
• reverse transcriptase
• DNA ligase
• Alkaline phosphatase
• terminal transferase
• Taq DNA polymerase
Restriction endonuclease
It can recognize special sequences
and cleave DNA at these specific
base sequences.
Type II can recognize palindrome
sequences.
GGATCC
CCTAGG
Palindrome
• Palindrome is also called inverted
repeat sequence, which means
the nucleotide sequence in 5′to
3′direction is the same in both
strands.
Sticky end and Blunt end
sticky ends
EcoRⅠ
5’…GAATTC…3’
3’…CTTAAG…5’
5’…G
3’…CTTAA
AATTC…3’
G…5’
PstⅠ
5’…CTGCAG…3’
3’…GACGTC…5’
5’…CTGCA
3’…G
G…3’
ACGTC…5’
blunt ends
Hae Ⅲ
5’…GGCC…3’
3’…CCGG…5’
5’…GG
3’…CC
CC…3’
GG…5’
Vector
• The term “vector” here refers to
some DNA molecules that can carry
a DNA fragment into a host cell for
replication.
• Including: plasmids, Bacteriophages
DNA, virus DNA ……
Vectors used in molecular cloning
Vector
(and host)
Plasmid
(bacteria, yeast)
Bacteriophage λ
(bacteria)
Cosmid
(bacteria)
Characteristics
Insert
size range
Small circular DNA
<5 - 10 kb
Linear viral DNA
up to ~20 kb
Hybrid of plasmid
and phage
up to ~50 kb
Yeast artificial
DNA containing yeast
chromosome (YAC) centromere, telomeres,
(yeast)
and origins of replication
~200 to
~1000 kb
plasmid
• Plasmids are small, circular molecules
of DNA that exist outside the main
bacterial chromosome and carry their
own genes for specialized functions.
Plasmid
4363bp
ori
Phage
• l phage DNA:
lgt phages: Insertion type vector
EMBL phages: replacement type vector
• M13 phage:
M13mp and pUC
EMBL phages
§2 Recombinant DNA
Technology
Process of cloning
• Isolation of target gene
• Selection and construction of
vectors
• Ligation of target DNA and vector
• Transformation of target gene into
receptor cell
• Screening for recombinant plasmids
• Expressing a cloned gene
Process of DNA cloning
§2.1 Isolation of target gene
1. Chemical synthesis
only for simple polypeptide chain
whose primary structure is clear.
2. Obtaining from genomic DNA library
3. Obtaining from cDNA library
4. polymerase chain reaction (PCR)
The genomic
DNA library is a
collection of the
comprehensive
DNA fragments
representing the
entire genome of
a species.
The cDNA library
represents the
population of
mRNAs, it only
contains the exons
of protein’s
structural genes.
mRNA
Reverse transcripase
cDNA
replication
dscDNA
vector
recombinate DNA
E. coli
recombinate DNA in E.coli
Preparation of cDNA library
Polymerase Chain Reaction
The polymerase chain reaction (PCR)
is a rapid and versatile in vitro
method for amplifying DNA.
PCR reaction system
•
•
•
•
•
DNA template
A pair of primers
DNA polymerase (Taq)
dNTPs
Mg2+-containing buffer
Procedures of PCR
• Denaturing: the template DNA is
denatured to become ssDNA from
dsDNA by heating.
• Annealing: this step allows the
hybridization of the primers with
target DNA.
• Extension: this process is the DNA
synthesis step.
ing
The first three
cycles of PCR
§2.2 Selection and construction of
vectors
A few commonly used vectors：
plasmid
phage
cosmid
yeast artificial chromosome (YAC)
§2.3 Ligation of target DNA and
vectors
1. Ligation of sticky end
GGATCC
CCTAGG
GGATCC
CCTAGG
GATCC
G
G
CCTAG
GATCC
G
G
CCTAG
DNA ligase
G GATCC
CCTAGG
2. Ligation of blunt ends
3. The addition of a homopolymer tail
4. Artificial linker
Adding a sequence of DNA fragment,
which contains the cleavage site for
restriction endonuclease.
Artificial linker
§2.4 Introduction of recombinant
DNA into recipient cell
• Introduction:
transformation
transfection
infection
Recipient cells
• Safe host bacteria
• Endonuclease and recombinase
deficiency
• Competent cells.
§2.5 Screening for recombinant
direct selection
• Screen of antibiotic resistance
markers
• Marker rescue (Insertion inactivation)
• In situ hybridization and
autoradiography
Antibiotic resistance genes
The procedure to form recombinant DNA
direct selection
Screen of antibiotic resistance markers
Marker rescue
In situ hybridization and
autoradiography
§2.6 Expression of the cloned gene
An expression vector is similar to
cloning vectors, but with a major
difference: the expression vector must
contain a promoter so that proteins can
be expressed.
Expression vector
Gene expression include:
• eukaryotic expression
• prokaryotic expression