13 recombinant dna technology

BIOTECHNOLOGY

any biology based technology which uses organisms or their parts to make or modify products or improve plants, animals and microorganisms.

RECOMBINANT DNA TECHNOLOGY

(Genetic Engineering, Molecular/Gene Cloning): coupling of techniques used in genetics with biological systems to achieve multiple goals in human, animal, plant, and microbial genetics as well as molecular pharming

recombinant DNA technology

A technique that involves isolating DNA fragments, inserting them into vectors, and replicating them in host cells such as bacteria or yeast.

reverse genetics

A method used to study gene function by introducing a mutated gene into an organism’s germ line.

restriction endonuclease

Endonucleases that recognize specific base sequences and break or restrict the DNA polymer at the sugar- phosphate backbone.

Originally isolated from bacteria where they function as a primitive defense system to cleave foreign DNA entering the bacterial cell.

Named after the organism from which they were isolated

BamH1

restriction endonuclease that was isolated from Bacillus amyloliquefaciens

Restriction endonuclease recognize short stretches of _____ (? bp) that contain specific nucleotide sequences.

dsDNA

4-8

Palindromes dsDNA

exhibits a 2-fold rotational symmetry.

Palindrome

when read in a 5'-3' direction the sequence at the top strand is identical to the bottom strand

Escherichia

genus: E

coli

Species: co

RY13

Strain: R

sticky ends

Staggered cuts/overhangs producing cohesive ends.

Results to ssDNA that are complementary to each other

blunt ends

Cuts straight in the middle

dsDNA

Do not form hydrogen bonds with each other

type I II III

major classes of restriction enzymes

type I

less common

Cut both strands at a nonspecific location > 1000 bp away from recognition site

3-subunit complex: individual recognition, endonuclease, & methylase activities

not useful in recombinant dna research

type II

most common

Cut both strands at a specific, usually palindromic, recognition site (4—8 bp)

Endonuclease and methylase are separate, single-subunit enzymes

very useful for recombinant dna research

type III

rare

Cleavage of one strand only, 24—26 downstream of the 3' recognition site

Endonuclease and methylase are separate 2-subunit complexes w/ one subunit in common

not useful in recombinant dna research

Human Genome Mapping Organization (HUGO)

agency founded in May 1989 to coordinate international research in Mapping Human genes (Human Genome Project)

Osteoarthritis

missense mutation replacing cysteine with arginine in the A-1 collagen gene

Aortic Aneurysm

missense mutation replacing glycine with arginine in the A1 collagen gene

Duchenne Muscular Dystrophy

deletion of the dystrophin gene

Huntington's Disease

extra copies of the CAG triplet (40-121 instead of the normal 7-25 copies) in the mRNA of the Huntington gene.

Fragile X syndrome

extra copies of the

CGG triplet (200-2000 instead of the

normal 6-50 copies) in the mRNA of the

Fragile X mental retardation gene (FMRI)

Breast Cancer

disposition gene (BRCA-I or BRCA-2) which interact with p53 gene

Numerous types of cancer is associated with problems of the p53 gene

Restriction Fragment Length Polymorphism (RFLP) analysis

DNA fingerprinting/profiling large amount of sample

Polymerase Chain Reaction (PCR)

DNA fingerprinting/profiling small amount of sample

research on animal genes

Super-ovulation hormone
transgenic fished
Transgenic Livestock and Poultry

research on plant genes

Transgenic plants

Mapping of the plant genome for various plant breeding applications

Potential bioterrorist agents

Yersinia pestis, Bacillus anthracis, Variola virus

National Institutes of Health (NIH)

developed guidelines regarding containment and handling of recombinant DNA molecules and organisms