Gene Cloning, Expression, and Mutagenesis

http://www.ncbi.nlm.nih.gov/books/

Type keywords: Molecular Cell Biology or DNA cloning and genomics

Central DogmaCentral Dogma

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Spliced RNASpliced RNA

ProteinProtein

GeneGene

TranscribedTranscribedRNARNA

5’ UTR5’ UTR 3’ UTR3’ UTRpApA

pApA

>< Splice acceptorSplice acceptor

Splice donorSplice donor

+1+1

+1+1

Definition of a gene: a unit of DNA contains the information to specify synthesis of a single polypeptide chain

DNA Cloning with Plasmid Vector (1)

DNA Cloning with Plasmid Vector (2)Cut and Paste

Cut-Restriction EnzymeAny enzyme that recognizes and cleaves a specific short sequence, the restriction site, in double-stranded DNA molecules. These enzymes are widespread in bacteria and are used extensively in recombinant DNA technology.

Paste- Ligation

DNA Cloning with Plasmid Vector (3)Cut and Paste

Source Microorganism Enzyme* Recognition Site Ends Produced

Arthrobacter luteus AluI AG   CT Blunt

Bacillus amyloliquefaciens H BamHI G   GATCC Sticky

Escherichia coli EcoRI G   AATTC Sticky

Examples

Ligation of Restriction Fragment with Cloning Vector

Polylinkers Facilitate Insertion of Restriction Fragments into Plasmid Vectors

Polylinker: a synthetic multiple-cloning-site sequence contains one copy of several restriction enzyme sites

Transformation

transformation Permanent, heritable alteration in a cell resulting from the uptake and incorporation of a foreign DNA. (Also, conversion of a "normal" mammalian cell into a cell with cancer-like properties usually induced by treatment with a virus or other cancer-causing agent.)

Screen Bacterial Colonies and Identify Your Correct Transformants

By using colony hybridizationBacterial colonies growing on agar plates can be transferred to nitrocellulose filters. The filters can be prepared for hybridization with your probe.

By using X-gal and IPTG: -complementationSome cloning vectors (pBluscript, pGEM..) contain coding region of -fragment of -galatosidase which is required for association with -fragment to form an active -galatosidase. Disruption of -fragment by inserting a foreign DNA causes inactivation of -galatosidase activity. (see next slide)

By random picking colonies- the most common methodYou may pick several colonies and isolate DNA using mini-prep method follow by restriction enzyme digestion to identify your correct colonies.

Identifying, Analyzing, and Sequencing Cloned DNA

Pick white colonies, mini-prep, restriction enzyme digestion

Confirm by sequencing

NCBI Blast search (Bioinformatics)

Screen Bacterial Colonies by Hybridization

3 Basic Cloning Step:

A.Prepare Target (cDNA or Genomic DNA)B.Prepare library of target DNA in a host/vector systemC.Screen library for sequence of interest

Prepare Genomic DNA and Construct a Genomic Library

Phage DNA isolation and subcloning into plasmid vector

Screen library for sequence of interest

How to select and label your probe?

How to select your probe? Subcloning of your cDNA or genomic DNA as probes Degenerated probes-Designing oligonucleotide probes based on protein sequence Obtain your cDNA clone (EST) in silicon other methods

Probe selection, labeling, and hybridization

How to label your probe? DNA end labeling

5’ end labelingFill-in end labeling

Labeling of a new DNA strandnick translation random primed labeling

Label your probe for hybridization DNA end labeling

5’ end labelingFill-in end labeling

Labeling of a new DNA strandnick translation random primed labeling

Labeling of RNARun-off in vitro transcription

linearization site

T3

In vitro transcription (RNA probe)

-32p-UTP

Phage DNA isolation and subcloning into plasmid vector

Pick white colonies, mini-prep, restriction enzyme digestion, southern blot analysis..

Confirm by sequencing

NCBI Blast search (Bioinformatics)

Southern Blot AnalysisThe technique of Southern blotting, named after its originator Edwin Southern, can identify specific restriction fragments in a complex mixture of restriction fragments. Technique for detecting specific DNA sequences separated by electrophoresis by hybridization to a labeled nucleic acid probe.

Sequencing (Sanger Method)

Bioinformatics

                                                                                        PubMed Entrez BLAST OMIM Books TaxBrowser Structure

  Search for Entrez GoNcbiHome

http://www.ncbi.nlm.nih.gov/

Add Reverse Transcription into Add Reverse Transcription into Central DogmaCentral Dogma

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Spliced RNASpliced RNA

ProteinProtein

GeneGene

TranscribedTranscribedRNARNA

5’ UTR5’ UTR 3’ UTR3’ UTRpApA

pApA

>< Splice acceptorSplice acceptor

Splice donorSplice donor

+1+1

+1+1

Definition of a gene: a unit of DNA contains the information to specify synthesis of a single polypeptide chain

Complementary DNA(cDNA)

Screen and Isolate cDNA clone

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GeneGene

Analyzing Cloned Gene

5’ UTR5’ UTR 3’ UTR3’ UTRpApA

+1+1

mRNAmRNA

Analyzing mRNA expression by northern blot method, in situ hybridization, RT-PCR, and DNA microarrays etc.

Determination of transcription start site by primer extension and S1 protection

Mapping the Transcription Start Site

S1 protection Primer extension

Expression Cloning

PCR Cloning

Producing high level of proteins from cloned cDNA

Note: E. coli lacks the enzymes that catalyze many of the post-translational modifications found on eukaryotic proteins. Thus, some eukaryotic proteins cannot be produced in active form in E. coli cells. This limitation can be overcome by developing eukaryotic expression system.

Expression of Cloned Gene

Baculovirus is a very large DNA virus (genome of about 150 kb) that infects insect cells. To express a foreign gene in baculovirus, the gene of interest is cloned in place of the viral coat-protein gene in a plasmid carrying a small part of the viral genome. The recombinant plasmid is cotransfected into insect cells with wild-type baculovirus DNA. At a low frequency, the plasmid and viral DNAs recombine through homologous sequences, resulting in the insertion of the foreign gene into the viral genome. Virus plaques develop, and the plaques containing recombinant virus look different because they lack the coat protein. The plaques with recombinant virus are picked and expanded. This virus stock is then used to infect a fresh culture of insect cells, resulting in high expression of the foreign protein.

Baculovirus Expression System

Translate Cloned cDNA In VitroRun-off in vitro transcriptionIn vitro trasnlation

linearization site

T3

In vitro transcription

In vitro translation

35S-MethionineRabbit reticulocyte lysate

In Vitro Mutagenesis

One of the most established techniques is site directed mutagenesis. By using this method, we can create mutations at any specific site in a gene whose wild-type sequence is already known. This known sequence is used to chemically synthesize short DNA segments called oligonucleotides. Through single-strand hybridization, these oligonucleotides can be directed to any chosen site in the gene. In one approach, the gene of interest is inserted into a single-stranded phage vector, such as the phage M13. A synthetic oligonucleotide containing the desired mutation is designed. This oligonucleotide is allowed to hybridize to the mutant site by complementary base pairing. The oligonucleotide serves as a primer for the in vitro synthesis of the complementary strand of the M13 vector.

Lac Operon