Chapter 4 Molecular Cloning Methods

Gene Cloning The Role of Restriction Endonuclease

Vectors Plasmids : carriers that allow replication of recombinant DNAs

r: resistant s: sensitive

lacZ’:  -galactosidase X-gal: synthetic substrate for  -galactosidase MCS: multiple cloning sites Blue/white color selection

Enhance of the cloning efficiency: dephosphorylation of the 5’-phophate in the vector by alkaline phosphatase to prevent self-ligation

Action of DNA ligase O P O O O CH 2 Base DNA ligase Restriction Enzyme O Base O OH P O O O CH 2 Base O O O _ _ _ 5’ 3’

Phages as Vectors replacement vectors * up to 20 kb of DNA can be cloned. * construction of genomic libraries * Charon 4: (12 kb< inserts <20 kb) Cosmids * cohesive ends of  phage DNA * plasmid origin of replication * up to kb of DNA can be cloned

M13 phage vectors * derived from filamentous phage M13 * contains lacZ’gene and MCS * DNA can be cloned in a single-strand form (introduction of site-specific mutation)

Phagemids * contains ori of filamentous phage f1, lacZ’gene and MCS * DNA can be cloned in a single-strand form via the help with f1 helper phage * contains T7 and T3 phage promoter for phage RNA polymerase (in vitro transcription to produce RNA transcripts)

Identifying a specific clone with a specific probe 1.Poly(oligo)nucleotide probes 2.Antibodies To probe the gene what we want: a.Homologous gene from another organism * Low/high stringency for hybridization of oligonucleotide probes to targets * High stringency: high temperature, high organic concentration, low salt concentration b. Degenerate primers from known amino acid sequence of target protein U G U UGG AUG UUC AAA AAC GA Trp Met Phe Lys Asn Glu

The Polymerase Chain Reaction (PCR) *Kary Mullis in the 1980s *Taq polymerase from Thermus aquaticus *Thermal cycler 95 o C 40 o C 72 o C 20x

Press Release 13 October 1993 The Royal Swedish Academy of SciencesThe Royal Swedish Academy of Sciences has decided to award the 1993 Nobel Prize in Chemistry for contributions to the development of methods within DNA-based chemistry, with half to Dr Kary B. Mullis, La Jolla, California, U.S.A., for his invention of the polymerase chain reaction (PCR) method, and half to Professor Michael Smith, University of British Columbia, Vancouver, Canada, for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies. Decisive progress in gene technology through two new methods: the polymerase chain reaction (PCR) method and site-directed mutagenesis The Nobel Prize in Chemistry 1993 Kary B. MullisMichael Smith

cDNA Cloning (complementary or copy DNA) Nick translation using E. Coli DNA polymerase I with 5’ 3’ exonuclease activity

Nick translation

Using RT-PCR in cDNA Cloning BamH1 HindIII

5’-RACE of a cDNA Rapid Amplification of cDNA Ends (completing of the 5’end of the mRNA) When we need to use 5’-RACE?

Methods of Expressing Cloned Genes Prokaryotic Expression Systems pUC and pBS vectors—lac promoter (in frame)

ptrpL 1 vector—trp operator/promoter (strong promoter) SD: Shine-Dargarno ribosome binding site

Expression of Hisx6-tagged fusion protein using pTrcHis vector EK: enterokinase (a protease) cutting site

Pellet B1 B2 M B1 B2 no-IPTG IPTG Sup M B1 B2 B1 B2 no-IPTG IPTG bigh3

CD109C SUP Pellet no-IPTG IPTG no-IPTG IPTG CI C2 C3 C1 C2 C3 M CI C2 C3 C1 C2 C3

Trx--His--S---- thrombinenterokinase

hr

rEKC1/201/40C1/201/40 4℃4℃ RT Each lane 含 25 λ sample (~5μg) Incubation time : 24hr

Expression of fusion protein using gt11 vector in E. Coli

Eukaryotic Expression Systems Expression in E.Coli: Inclusion bodies No posttranslational modification Expression in yeast Expression in caterpillar using Baculovirus-derived vectors (10% in dry mass, polyhedrin)

( 中原大學生物科技學系吳宗遠教授 ) Baculovirus

Real-Time PCR Real-time PCR quantifies the amplification of the DNA as it occurs As DNA strands separate, anneal to forward and reverse primers, and to fluorescent-tagged oligonucleotide complementary to part of one DNA strand

Fluorescent Tags in Real-Time PCR This fluorescent-tagged oligonucleotide serves as a reporter probe –Fluorescent tag at 5’-end –Fluorescence quenching tag at 3’-end With PCR rounds the 5’ tag is separated from the 3’ tag Fluorescence increases with incorporation into DNA product

Real-Time PCR --- RT-PCR Reverse-transcription PCR --- RT-PCR Quantitative real-Time PCR --- QRT-PCR, qRT-PCR

Real-time polymerase chain reaction Quantitative real time polymerase chain reaction (QRT-PCR) Kinetic polymerase chain reaction A process used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample (1) Real-time PCR using double-stranded DNA dyes (2) Fluorescent reporter probe method

(1) Real-time PCR using double-stranded DNA dyes

Fig. 4.17

TaqMan assay (named after Taq DNA polymerase) was one of the earliest methods introduced for real time PCR reaction monitoring and has been widely adopted for both the quantification of mRNAs and for detecting variation. The method exploits the 5' endonuclease activity of Taq DNA polymerase to cleave an oligonucleotide probe during PCR, thereby generating a detectable signal.DNA polymerase (2) Fluorescent reporter probe method