Genetic Recombination and Genetic Engineering

Recombination activating gene enzyme RAG1 and RAG2 CACAGTG 12/23 ACAAAAACC GTGTCAC TGTTTTTGG RSS Recombination signal sequence RSS... insertion sequences IS including: inverted repeats

Section 1

DNA Recombination

§1.1 homologous Recombination

• Homologous recombination occurs b etween identical or nearly identical s equences It is also called general re combination.

DNA invading

(recA)

Branch migration (recA)

DNA ligase

5´

3´

5´ 3´

endonuclease (recBCD)

endonuclease (recBCD)

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

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

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 sp

ecific pieces of DNA in the genome.

• Transposition resembles site-specifi

c recombination being catalyzed by s pecial 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

• Insertion sequence + another

gene (usually antibiotic gene)

Transposase gene tet-R gene

Transposons Transposition

Section 2 Recombinant DNA

Technology

Clone

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

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

It can recognize special sequences and cleave DNA at these specific b ase sequences.

Type II can recognize palindrome s equences

Restriction endonuclease

• Palindrome is also called inverted

the nucleotide sequence in 5′to

3′direction is the same in both

strands.

• The term “ vector ” here refers to som

e DNA molecules that can carry a DN

A fragment into a host cell for replica tion.

• Including: plasmids , Bacteriophages DNA, virus DNA ……

Vectors used in molecular cloning

Yeast artificial DNA containing yeast ~200 to

chromosome (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

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 vector s

• Ligation of target DNA and vector

• Transformation of target gene into re ceptor cell

• Screening for recombinant plasmids

Process of cloning

Process of DNA cloning

2 Obtaining from genomic DNA library

3 Obtaining from cDNA library

4 polymerase chain reaction (PCR)

The cDNA library rep

resents the populati

Preparation of cDNA library

Polymerase Chain Reaction

　The polymerase chain reaction (PCR)

is a rapid and versatile in vitro metho

d for amplifying DNA

ing

A few commonly used vectors ：

plasmid phage cosmid yeast artificial chromosome (YAC)

§2.2 Selection and construction of

vectors

GGATCC CCTAGG

GGATCC CCTAGG

G CCTAG

GATCC

G

G CCTAG

2 Ligation of blunt ends

3 The addition of a homopolymer tail

Adding a sequence of DNA fragment , which contains the cleavage site for re striction endonuclease.

4 Artificial linker

Artificial linker

• 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

direct selection

Antibiotic resistance genes

direct selection

The procedure to form recombinant DNA

Screen of antibiotic resistance markers

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: