Gene Cloning, Expression, and Mutagenesis Type keywords: Molecular Cell Biology or DNA cloning and genomics.
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Gene Cloning, Expression, and Mutagenesis http://www.ncbi.nlm.nih.gov/books/ Type keywords: Molecular Cell Biology or DNA cloning and genomics
Central Dogma <<<<> >> > ATGstop Spliced RNA Protein Gene TranscribedRNA 5’ UTR 3’ UTR pA pA > < Splice acceptor Splice donor +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 (2) Cut and Paste Cut-Restriction Enzyme Any 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 MicroorganismEnzyme*Recognition SiteEnds Produced Arthrobacter luteusAluIAG CT Blunt Bacillus amyloliquefaciens HBamHIG GATCCSticky Escherichia coliEcoRIG AATTCSticky 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 hybridization Bacterial 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: -complementation Some 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 method You may pick several colonies and isolate DNA using mini-prep method follow by restriction enzyme digestion to identify your correct colonies.
3 Basic Cloning Step: A.Prepare Target (cDNA or Genomic DNA) B.Prepare library of target DNA in a host/vector system C.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 labeling Fill-in end labeling Labeling of a new DNA strand nick translation random primed labeling
Label your probe for hybridization DNA end labeling 5’ end labeling Fill-in end labeling Labeling of a new DNA strand nick translation random primed labeling
Labeling of RNA Run-off in vitro transcription linearization site T3 In vitro transcription (RNA probe) - 32 p-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 Analysis The technique of Southern blotting, named after its originator Edwin Southern, can identify specific restriction fragments in a complex mixture of restriction fragments.Southern blotting, Technique for detecting specific DNA sequences separated by electrophoresis by hybridization to a labeled nucleic acid probe.probe.
Bioinformatics PubMedEntrezBLASTOMIMBooksTaxBrowserStructure Search for http://www.ncbi.nlm.nih.gov/
Add Reverse Transcription into Central Dogma <<<<> >> > ATGstop Spliced RNA Protein Gene TranscribedRNA 5’ UTR 3’ UTR pA pA > < Splice acceptor Splice donor +1 +1 Definition of a gene: a unit of DNA contains the information to specify synthesis of a single polypeptide chain Complementary DNA (cDNA)
<<<<> >> > ATGstop Gene Analyzing Cloned Gene 5’ UTR 3’ UTR pA +1 mRNA 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 protectionPrimer extension
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 Vitro Run-off in vitro transcription In vitro trasnlation linearization site T3 In vitro transcription In vitro translation 35 S-Methionine Rabbit 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.