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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. - PowerPoint PPT Presentation
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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.
DNA invading(recA)
Branch migration
(recA)
DNA ligase
5´ 3´
5´3´5´
3´
5´3´
5´ 3´
5´ 3´
5´3´
5´3´
5´ 3´
5´ 3´
5´3´5´3´
5´3´
5´ 3´
5´ 3´
5´3´
5´3´
3´5´
5´ 3´
5´3´
5´3´
Holiday intermediate
5´ 3´
5´ 3´
5´3´5´3´
endonuclease
(recBCD)
endonuclease
(recBCD)
5´ 3´
5´ 3´
5´3´5´3´
5´
3´
5´
3´
5´
3´
5´3´
Holliday intermediate
5´3´
5´
5´5´
3´
3´
3´
5´ 5´
5´5´
3´
3´
3´
3´5´3´
5´
5´5´
3´
3´
3´
5´ 5´
5´5´
3´
3´
3´
3´5´3´
5´
5´5´
3´
3´
3´
endonuclease
(ruvC)
endonuclease
(ruvC)
DNA ligase DNA ligase
patch recombinant
splice recombinant
• Bacterial Conjugation has been defined
as the transmission of genetic information
from a donor bacterium to a recipient cell
through cell-to-cell contact.
§1.2 Conjugation
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.
DNA
Transformation
Transformation experiment of Strept
ococcus pneumoniae
§1.4 Transduction
• Transduction is the transfer of DNA
fragments from one bacterium to an
other bacterium by a bacteriophage.
Transduction
• Site-specific recombination occurs at a specific DNA sequence.
• The first example was found in the integration between DNA and E. coli DNA.
§1.5 Site-specific Recombination
λDNA integration
P1 H1P2hin H2 ×è¶ô»ùÒò
DNA
P1 H1
P2
hin H2 ×è¶ô»ùÒò
H segment H1 flagellin
H2 flagellin
repressor
P2
P2
hix hix
Phase variation of Salmonella typhimurium flagella
Hin
rH1
rH1
Recombination activating gene enzyme
(RAG1 and RAG2)
CACAGTG (12/23) ACAAAAACC
GTGTCAC TGTTTTTGG
RSS
Recombination signal sequence (RSS)
§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.
insertion sequences (IS) including:
inverted repeats (IR) : 9~41bp
transposase gene
repeated sequences : 4~12bp
IS Transposition
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
Clone
A clone is defined as a number of ident
ical copy (molecules, cells or individua
ls) all derived from a common ancestor.
Also named asexual multiplication.
§2.1 Correlative concepts
DNA Cloning
DNA cloning involves separating a spe
cific gene or segment of DNA from its l
arger chromosome and attaching it to
a small molecule of carrier DNA, then r
eplicating this modified DNA thousand
s 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.
• restriction endonucleases
• DNA polymeraseⅠ• reverse transcriptase
• DNA ligase
• Alkaline phosphatase
• terminal transferase
• Taq DNA polymerase
Applications in enzymology
It can recognize special sequences and cleave DNA at these specific base sequences.
Type II can recognize palindrome sequences.
Restriction endonuclease
GGGGAATTCCCCCCCCTTAAGGGG
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 ends
EcoRⅠ 5’…GAATTC…3’ 5’…G AATTC…3’3’…CTTAAG…5’ 3’…CTTAA G…5’
PstⅠ 5’…CTGCAG…3’ 5’…CTGCA G…3’3’…GACGTC…5’ 3’…G ACGTC…5’
blunt ends
Hae Ⅲ 5’…GGCC…3’ 5’…GG CC…3’3’…CCGG…5’ 3’…CC GG…5’
Sticky end and Blunt end
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 Insert (and host) Characteristics size rang
e
Plasmid Small circular DNA <5 - 10 kb (bacteria, yeast)
Bacteriophage λ Linear viral DNA up to ~20 kb (bacteria)
Cosmid Hybrid of plasmid up to ~50 kb (bacteria) and phage
Yeast artificial DNA containing yeast ~200 tochromosome (YAC) centromere, telomeres, ~1000 kb (yeast) and origins of replication
plasmid
• Plasmids are small, circular molecules
of DNA that exist outside the main
bacterial chromosome and carry their
own genes for specialized functions.
Plasmid
ori
4363bp
Phage
• phage DNA:
gt phages: Insertion type vector
EMBL phages: replacement type vector
• M13 phage:
M13mp and pUC
EMBL phages
§2 Recombinant DNA Technology
• 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 cloning
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 library4. 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
A few commonly used vectors :
plasmid
phage
cosmid
yeast artificial chromosome (YAC)
§2.2 Selection and construction of vectors
GGATCCCCTAGG
GGATCCCCTAGG
GCCTAG
GATCCG
GCCTAG
GATCCG
DNA ligase
GCCTAG
GATCCG
§2.3 Ligation of target DNA and vectors
1. Ligation of sticky end
2. Ligation of blunt ends
3. The addition of a homopolymer tail
Adding a sequence of DNA fragment, which contains the cleavage site for restriction endonuclease.
4. Artificial linker
Artificial linker
§2.4 Introduction of recombinant
DNA into recipient cell
• Introduction:
transformation
transfection
infection
• Safe host bacteria
• Endonuclease and recombinase defi
ciency
• Competent cells.
Recipient cells
§2.5 Screening for recombinant
• Screen of antibiotic resistance markers
• Marker rescue (Insertion inactivation)
• In situ hybridization and
autoradiography
direct selection
Antibiotic resistance genes
direct selection
The procedure to form recombinant DNA
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 clonin
g vectors, but with a major difference: th
e expression vector must contain a pro
moter so that proteins can be expressed.
Expression vector
• eukaryotic expression
• prokaryotic expression
Gene expression include: