cloning

Methods of Molecular Biology: 

Methods of Molecular Biology

2.1 Obtain and Express a Gene: 

2.1 Obtain and Express a Gene Methods to amplify a gene, including cDNA. Vectors to clone a gene. Vectors to express a gene.

2.1.1 Gene Cloning: 

2.1.1 Gene Cloning

The Role of Restriction Endonucleases: 

The Role of Restriction Endonucleases

Name and cutting sites: 

Name and cutting sites H aemophilus in fluenae strain R d : Hin dII 4 4 =256,4 6 =4096,4 8 =65000(rare cutters) Sma I & Xma I: Identical recognition sequences but different cutting sites, called Isoschizomers.

Recombination with restriction endonuclease and DNA ligase: 

Recombination with restriction endonuclease and DNA ligase

An example of vectors: 

An example of vectors

Cloning and screening: 

Cloning and screening Replica plating

Convenient cloning vectors: 

Convenient cloning vectors With MCS: Multiple cloning site; With α-complementation: α-peptide encoded by lacZ’, and ω-peptide encoded by the host. An active β-glacto’sidase can turn X-gal into blue.

λ-phage vectors: 

λ-phage vectors Replacement vector: the region in the middle of phage DNA is taken out, with two arms left. That can not affect replication of DNA. Suitable for genomic libraries, with a capacity of up to 20kb. Note that target DNA is only incomplete digested with Eco RI, to yield 16-20kb fragments.

Screening with plaque hybridization: 

Screening with plaque hybridization In λgtll, Cloned genes are put under the control of a lac promoter, which mean the transcription and translation of this gene can occur.

M13 phage vectors: 

M13 phage vectors Replicative Form The Genome of M13 phage is a single stranded DNA. So DNA fragment cloned into this vector can be recovered in single stranded form. Single stranded DNA can be aid to site directed mutagenesis.

2.1.2 PCR-Polymerase Chain Reaction: 

2.1.2 PCR-Polymerase Chain Reaction

Basic procedure of PCR: 

Basic procedure of PCR Denaturation: 95℃ Annealing: 55℃ Enlongation: 72℃ Suppose we repeat the cycle, we will get 2 30. An efficient way to amplify a gene even though the whole sequence is not totally clear.

cDNA cloning: 

cDNA cloning cDNA is short for complementary DNA or copy DNA. Sometimes we want to make a cDAN library, a set of clones representing as many as possible of the mRNAs in a given time or a specific cell. Sometimes we want to study one special mRNA, especially in an eukaryotic cell.

Making a cDNA library: 

Making a cDNA library Nick tranlation

Nick Translation: 

Nick Translation

RT-PCR for cloning of a specific cDNA: 

RT-PCR for cloning of a specific cDNA Hin dIII site Bam HI site Hin dIII site Bam HI site Primers here are called GSPs.

1. One can use plasmid vectors such as pUC to clone cDNA, and positive clones can be identified by clonoy hybtidization; 2. Or one can use λ phage vectors, such as λgtll, and identify target gene by plaque hybridization.: 

1. One can use plasmid vectors such as pUC to clone cDNA, and positive clones can be identified by clonoy hybtidization; 2. Or one can use λ phage vectors, such as λgtll, and identify target gene by plaque hybridization.

Rapid Amplification of cDNA ends: 

Rapid Amplification of cDNA ends For whatever reason, very frequently within cDNA library, a cDNA is not full length, usually the 5’end is missing. A special technique call RACE (Rapid Amplification of cDNA ends) can be used to fill the incomplete end.

Procedure of 5’ RACE: 

Procedure of 5’ RACE Note that cDNAs are double stranded in cDNA library, and 5’ RACE is for filling the missing 5’ end of a coding strand

2.1.3 Methods of Expressing Cloned Genes: 

2.1.3 Methods of Expressing Cloned Genes

Bacterial Expression vectors: 

Bacterial Expression vectors Bacterial expression vectors typically have two elements that are required for active gene expression: a strong promoter and a ribosome binding site (RBS, SD sequence) near an initiating ATG codon Usually expression vectors should be inducible, otherwise growth of bacterial hosts can be largely affected, and inclusion bodies could be produced.

Producing a fusion protein by a pUC vector: 

Producing a fusion protein by a pUC vector A lac promoter can be induced by isopropylthioglactoside (IPTG).

Shortcomings of lac promoter: 

Shortcomings of lac promoter Lac promoter can be leaky. Expressing a gene in vectors with their own lac Is (like pBS) or using a T7 promoter in plasmid to control, which is also in high efficiency. Lac promoter is not strong enough. We can use trc promoter. It’s a hybrid promoter with the -35 box of trp promoter which is very strong and with -10 box of lac promoter which can be induced by IPTG.

pTrc-His: 

pTrc-His

Expression and Purification: 

Expression and Purification Nickel affinity chromatography

Synthesizing a fusion protein in λ gt11: 

Synthesizing a fusion protein in λ gt11 Popular for making and screening cDNA library directly by antiserum (with labeled protein A which binds to most antibodies).

Detecting positive λ gt11 clones by antibody screening: 

Detecting positive λ gt11 clones by antibody screening

Eukaryotic Expression Systems: 

Eukaryotic Expression Systems E. coli cells sometimes destroy expression products of cloned eukaryotic genes. Prokaryotes do not carry out the same posttranslational modification as eukaryotes do. Interior of bacteria is not as conducive to proper folding of eukaryotic proteins as the interior of eukaryotes.

Examples: 

Examples Yeast shuttle vectors are based on 2-micro plasmid inhabiting in yeast, also contain pBR322 replication origin Baculovirus vector: derived from baculovirus that infects the caterpillar . Viruses in this class have a rather large circular DNA genome. (have details)

Details: 

Powerful polyhedrin promoter. Polyhedrin ( 多角体蛋白 ) is a structural protein of baculovirus which should be expressed with copious quantity in infected cells. 15 time lager than transfer vector, not suitable for cloning of target gene and replication in bacterial host. Details

How to do transfection: 

How to do transfection Transformation means the conversion of a normal cell to a cancer-like cell. 1. cells take up DNA together with Ca 3 (PO 4 ) 2 crystal. 2. liposomes containing DNA can fuse with cell membranes. 3. plant cells are commonly transfected by a biolistic method( 基因枪 ).

2.2 Molecular Separations: 

2.2 Molecular Separations

2.2.1 Electrophoresis: 

2.2.1 Electrophoresis From cathode to anode

DNA Gel Electrophoresis: 

DNA Gel Electrophoresis EB fluoresces orange under ultraviolet light.

Analysis of DNA size by gel electrophoresis: 

Analysis of DNA size by gel electrophoresis Vertical axis is logarithmic. Electrophoresis mobility (migration rate) is inversely proportional to the log of size. Limited for very large DNA

Pulsed-field gel electrophoresis (PFGE) to analyze large DNAs even in chromosomes: 

Pulsed-field gel electrophoresis (PFGE) to analyze large DNAs even in chromosomes 2.2Mb 0.2Mb

SDS-PAGE to analyze protein molecules: 

SDS-PAGE to analyze protein molecules Prestained Markers. Usually stained with Coomassie Blue. SDS: Sodium Dodecyl Sulfate PAGE: PolyAcrylamide Gel Electrophesis

Two dimensional gel electrophoresis: 

Two dimensional gel electrophoresis No benzoic acid With benzoic acid E. coli

Why two dimension?: 

Why two dimension? First dimension: Isoelectric focusing, with a pH gradient. For first dimension, from cathode to anode, what would the pH gradient be like? Second dimension: SDS-PAGE.

2.2.2 Chromatography: 

2.2.2 Chromatography Ion-Exchange Chromatography Anion-exchange: resin can bind anion. Cation-exchange: resin can bind cation. Beads Void volume Who escape first?