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 Streptococcus pneumoniae

　§1.4 Transduction

• Transduction is the transfer of DNA fragments from one bacterium to another 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 TGTTTTTGGRSS

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) ： 9~41bptransposase generepeated 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 identical copy (molecules, cells or individuals) all derived from a common ancestor. Also named asexual multiplication.

§2.1 Correlative concepts

　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.

　• 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 vector

s• Ligation of target DNA and vector• Transformation of target gene into re

ceptor cell• Screening for recombinant plasmids• Expressing a cloned gene 　

Process of cloning

　Process of DNA cloning

　§2.1 Isolation of target gene

1. Chemical synthesisonly for simple polypeptide chai

n whose primary structure is clear.

2. Obtaining from genomic DNA library3. 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.

mRNAReverse transcripase

cDNA

replication

dscDNA

vector

recombinate DNAE. 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 ：

plasmidphagecosmid 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 cloning vectors, but with a major difference: the expression vector must contain a promoter so that proteins can be expressed.

Expression vector

• eukaryotic expression

• prokaryotic expression

Gene expression include: