Foundations of Biology Chapter 20

recombinant DNA technology

The use of laboratory techniques to bring together DNA fragments from two or more sources.

cDNA (complementary DNA)

A DNA molecule made in vitro using mRNA as a template and the enzyme reverse transcriptase; lacks introns

DNA cloning

Process of creating numerous copies of a gene of interest

plasmid

A vector consisting of a circular strand of DNA that is commonly found in bacteria

vector

A DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell

recombinant

Describes an organism or genome which contains DNA from an outside source

transformant

Describes an organism which has successfully taken up a vector, such as a plasmid

restriction enzyme

An enzyme that recognizes particular DNA sequences and cleaves the DNA backbone at two sites

sticky ends

Single stranded ends of DNA fragments that will hydrogen bond to each other due to their complementary sequences

blunt ends

Both strands terminate in a base pair; no single stranded regions are exposed

DNA ligase

An enzyme which joins nucleotides together via covalent bonds

transformation

The genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from the surroundings

DNA library

A collection of transformed bacteria which each harbor a plasmid with an inserted DNA fragment

cDNA library

A DNA library made from mRNA that includes clones of highly expressed genes only

genomic library

A DNA library made using the entire genome which includes the entire gene with introns; almost all clones that are expressed in the tissue of origin will be found here

probe

A fragment of DNA or RNA of variable length which can be radioactively labeled that is used to detect a complementary target nucleotide sequences

biotechnology

The use of living organisms or the products of living organisms for human benefit

polymerase chain reaction (PCR)

A technique used to make many copies of a gene in vitro; uses primers to flank a DNA region and amplify it

primer

A short segment of RNA required to begin DNA replication

dNTPs

A nucleotide with three phosphate groups that can be used as an energy source

phosphatase

An enzyme which removes phosphate groups from proteins

blue white screening

A screening technique that allows for the rapid and convenient detection of recombinant bacteria. Cells are grown in X-gal and the color of the cells indicates whether or not transformed cells because the gene of interest is inserted into the lacZ gene

Blue

This color in blue white screening indicates that the gene of interest was not inserted into the plasmid

White

This color in blue white screening indicates that the gene of interest was inserted into the plasmid

multiple cloning site

A site on a plasmid that contains many unique restriction sites which allows for the precise insertion of DNA into the vector

origin of replication

A site within a chromosome that serves as the starting point for DNA replication

selectable marker

A gene whose presence can allow organisms to grow under a certain set of conditions. Ex. antibiotic resistance

competent cells

Cells whose cell walls have been altered to allow DNA to pass through more easily

heat shock

Technique used to make cells uptake DNA by exposing them to high temperatures

electroporate

Technique used to make cells uptake DNA by exposing them to an electric field

Taq polymerase

A thermostable DNA polymerase used in PCR

denaturing

Step in PCR which occurs around 96 degrees celsius; DNA strands are separated using heat

annealing

Step in PCR which occurs between 55 and 65 degrees celsius; primers bind to DNA

extension

Step in PCR which occurs around 72 degrees celsius; taq polymerase synthesizes DNA from the primers

RT-PCR

PCR variant which detects gene expression through the creation of cDNA transcripts via reverse transcriptase; used to diagnose genetic diseases

gene therapy

The introduction of healthy alleles to replace or augment existing alleles in order to fight disease

genetic engineering

The direct manipulation of genes for practical purposes

retrovirus

An RNA virus that uses reverse transcription to produce viral DNA that is integrated into the host cell's genome

reverse transcriptase

An enzyme that catalyzes the synthesis of DNA starting with RNA as a template

virion

A complete virus particle that consists of an RNA or DNA core with a protein coat sometimes with external envelopes and that is the extracellular infective form of a virus

tissue tropism

The cells and tissues of a host that support growth of a particular virus or bacterium

pseudotyping

The process of producing viruses or viral vectors in combination with foreign viral envelope proteins.

replication incompetent virus

A virus which lacks coding regions for addition rounds of virion replication

packaging cell line

Cells which produce retroviruses

Agrobacterium

Bacteria used to transform plant cells

crown gall tumor

A tumor generated by agrobacterium

Ti plasmid

Tumor inducing plasmid found in Agrobacterium that an be used as a cloning vector

T-DNA

DNA that is transferred from a Ti plasmid into a host plant's nuclear genome

severe combined immunodeficiency (SCID)

A rare genetic disorder that makes patients susceptible to infectious diseases because their immune systems are severely compromised

golden rice

A GM rice variant which has beta carotene which is used to fight vitamin A deficiency

Ex vivo

Cells are extracted, altered, and then put back into the body

zinc finger nuclease

Artificial restriction enzyme that targets unique sequences in the genome and can be used to alter genomes by taking advantage of DNA repair machinery through the use of zinc finger DNA binding domains

TALENs

A set of restriction enzymes that cut specific sequences of DNA using TAL effector DNA binding domaisn

transgenic

Describes an organism that carries genes that were introduced using molecular techniques

1. Isolate gene of interest (GoI)

2. Choose a vector

3. Cut DNA with restriction enzyme

4. Ligate DNA into vector

5. Transform vector into cells

6. Select transformants

7. Select recombinants

What are the 7 steps of gene cloning and transformation?

Antibodies

Proteins involved in immune response that can be found in the blood and lymphatic fluid of mammals. They all have the similar basic structure of two light chain polypeptides bonded to two heavy chain polypeptides.

Fab component

Where antibody binds to antigen

Fe region

The effector region of an antibody

Western blot

Test which uses gel electrophoresis and antibodies in order to detect and analyze specific proteins. Proteins are separated by molecular weight then exposed to antibodies and banding patterns are observed

Co-immunoprecipitation

A technique to determine whether two or more proteins are associated with each other, in which one protein is isolated by binding to a specific antibody and any associated proteins are identified in the immunoprecipitate, e.g. by Western blotting.

Immunofluorescence

Method of tagging antibodies with a luminating dye to detect antigen-antibody complexes