Gene Cloning, Expression, and Mutagenesis http://www.ncbi.nlm.nih.gov/books/ Type keywords: Molecular Cell Biology or DNA cloning and genomics
> Splice donor < Splice acceptor Central Dogma > < < < > < > > Gene ATG stop Transcribed RNA pA +1 5’ UTR 3’ UTR Spliced RNA pA +1 Protein 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 Examples
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 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. • By using X-gal and IPTG: a-complementation Some cloning vectors (pBluscript, pGEM..) contain coding region of a-fragment of b-galatosidase which is required for association with w-fragment to form an active b-galatosidase. Disruption of a-fragment by inserting a foreign DNA causes inactivation of b-galatosidase activity. (see next slide) • 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.
Identifying, Analyzing, and Sequencing Cloned DNA Pick white colonies, mini-prep, restriction enzyme digestion Confirm by sequencing NCBI Blast search (Bioinformatics)
3 Basic Cloning Step: • Prepare Target (cDNA or Genomic DNA) • Prepare library of target DNA in a host/vector system • Screen library for sequence of interest
Screen library for sequence of interest How to select and label your probe? Phage DNA isolation and subcloning into plasmid vector
Probe selection, labeling, and hybridization • 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 • 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 a-32p-UTP In vitro transcription (RNA probe) T3
Pick white colonies, mini-prep, restriction enzyme digestion, southern blot analysis.. Confirm by sequencing NCBI Blast search (Bioinformatics) Phage DNA isolation and subcloning into plasmid vector
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. Technique for detecting specific DNA sequences separated by electrophoresis by hybridization to a labeled nucleic acid probe.
Sequencing (Sanger Method)
> Splice donor < Splice acceptor Add Reverse Transcription into Central Dogma > < < < > < > > Gene ATG stop Transcribed RNA pA +1 5’ UTR 3’ UTR Spliced RNA Complementary DNA (cDNA) pA +1 Protein Definition of a gene: a unit of DNA contains the information to specify synthesis of a single polypeptide chain
Analyzing Cloned Gene > < < < > < > > Gene ATG stop 5’ UTR 3’ UTR mRNA pA +1 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 of Cloned Gene 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.
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 In vitro transcription 35S-Methionine Rabbit reticulocyte lysate T3 In vitro translation
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.