Recombinant DNA

Recombinant DNA

Joining of molecules (usually artificially to biological sources not found in nature)

Technology: used to isolate replicate and analyze genes

Clones

Recovered copies of recombinant DNA used to study structure and orientation of DNA

Restriction enzymes

DNA cutting enzyme. Binds ot DNA at specific recognition sequence (recognition site) and cleace DNA to produce restriction fragments. Enzyme cleaves with strands of DNA (digestion).

Produced by bacteria as defense mechanism against bacteriaphage.

1. Sticky (cohesive) ends: fragments produced with overhang

2. Blunt ends: fragments produced with double stranded ends

DNA ligase

DNA fragments will seal phosphodiester backbone. Joins restriction fragments covalently to produce intact DNA molecules

Vector

Carries of DNA molecules.

1. Can replicate cloned DNA fragments in host cells

2. Must be able to replicate independently

3. Have several restriction enzyme sites to allow insertion of DNA fragments

4. Carry selectable gene marker to distinguish host cells that have taken them up from those that have not

Plasmids used in DNA cloning

Genetically modified bacterial plasmid. (A number of convenient restriction sites and a marker gene to select for presence in host cell)

Plasmids introduction to bacteria via transformation.

1. Using calcium ions and brief heat shock to pulse DNA into cells.

2. Electroporarion: a brief high intensity pulse of electricity to move DNA into bacterial cells

Multiple cloning sites

1. Plasmid DNA and DNA to be cloned are cut with the same restriction enzyme.

2. DNA restriction fragments from DNA to be cloned are added to linearized vector in presence of DNA ligate.

3. Recombinant DNA is produced and introduced into bacterial host cells by transformation

Selectable marker gene

Genes provide resistance to antibodies

Blue white selection: used to identify cells containing recombinant and nonrecombinant DNA

Blue white screening mechanism: agar plate containing X-gal (analog of lactose; substrate for Ɓ-galactosidase). When X-gal is cleaved by enzyme it turns Blue. (Bacterial cells with functional lacZ gene carrying a nonrecombinant plasmid=Blue. Bacterial cells with recombinant plasmid=white)

BACs and YACs

BAC: bacterial artifical chromosome, generally large but low copy number plasmid

YAC: yeast artifical chromosome have telomeres at each end, origin of replication, and centromere

Expression vector

Designed to ensure mRNA expression of cloned gene to produce large quantities of encoded proteins in host cells

DNA libraries

Represent a collection of cloned DNA.

1. Genomic: contains at least one copy of all sequences in genome of interest. Constructed by cutting genomic DNA with restriction enzyme and ligating fragments into vectors

2. Complementary: contains complementary DNA copies made from mRNAs present in cell population. Represents genes active transcriptionally at the time cells were collected for mRNA isolation. (Isolating mRNA from cells, Synthesizing DNA using reverse transcriptase, Cloning cDNA molecules into vectors).

Reverse transcriptase

Used to generate cDNA from mRNA

Extends oligo(dT) primer and synthesizes complementary DNA copy of mRNA

Produces mRNA-DNA double stranded hybrid molecule

Library screening

Used to sort through libraries and isolate specific genes of interest.

1. Clones from library are grown on agar plate to produce colonies

2. Colonies are screened by transferring bacterial colonies from plate to filter

3. Filter is hybridized with nucleic acid probe to DNA sequence of interest

4. Colony corresponding to probe identified on filter is recovered

5. Phase library is screened by plaque hybrization

6. Libraries enable scientists to clone DNA and identify individual genes in library

Probe

Any DNA/RNA sequence complementary to target gene of sequence being identified. Used to screen libraries and recover clones of specific genes