Genetic Recombination and Genetic Engineering Chapter 14 Genetic Recombination and Genetic Engineering

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 endonuclease DNA invading (recA) endonuclease ligase 5´ 3´ 5´ 3´ endonuclease (recBCD) DNA invading (recA) 5´ 3´ Branch migration (recA) 3´ 5´ endonuclease (recBCD) 5´ 3´ DNA ligase 5´ 3´ Holiday intermediate

Holliday intermediate 5´ 3´ 5´ 3´

DNA ligase DNA ligase splice recombinant patch recombinant 3´ 5´ endonuclease (ruvC) 5´ endonuclease (ruvC) 3´ 3´ 5´ 5´ 3´ 5´ 5´ 3´ 3´ DNA ligase DNA ligase 5´ 3´ 5´ 3´ splice recombinant patch recombinant

§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

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 P2 rH1 H2 flagellin Hin repressor P2 rH1 H segment 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’ 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’

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 range 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 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 ori

Phage  phage DNA: gt 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.

recombinate DNA in E.coli mRNA The cDNA library represents the population of mRNAs, it only contains the exons of protein’s structural genes. 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

3. The addition of a homopolymer tail 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 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