8. Recombinant DNA Technology (1)

what is recombinant dna technology

intentionally modifying genomes of organisms for practical purposes

3 goals of recombinant technology

eliminate undesirable phenotypic traits

combine traits of two or more organisms

create organisms that synthesize products humans need

what are mutagens

physical and chemical agents that produce mutations

how do scientist use mutagens

create changes in microbes genomes to change phenotypes

select for and culture cells with beneficial characteristics

the use of reverse transcriptase to synthesize cDNA

isolated from retroviruse

uses rna template to transcribe molecule of dna

easier to isolate mRNA molecule for desired protein first

mrna of eukaryotes has introns removed

synthetic nucleic acids

molecules of dna and rna produced in cell free solutions

uses of synthetic nucleic acids

elucidating genetic code

creating genes for specific protiens

synthesizing dna and rna probes to located sequences of nucleotides

synthesizing antisense nucleic acid molecules

restriction enzymes

bacterial enzymes that cut dna molecules at restriction sites

-cuts with sticky ends

-cuts with blunt ends

vectors

nucleic acid molecules that deliver a gene into a cell

useful properties of vectors

small enough to manipulate in labs

survive inside cells

recognizable genetic marker

genetic expression of gene

viral genomes, transposons, plasmids

what are gene libraries

collection of bacterial or phage clones where each clone contains one gene of an organisms genome

-may contain all genes of a single chromosome

-may contain set of cDNA complementary to mRNA

multiplying dna in vitro: Polymerase chain reaction

large number of identical molecules of DNA produced in vitro

critical to amplify DNA in variety of situations

repetitive process of multiplying dna: PCR

denaturation

priming

extension

*automated using a thermocycler*

how to select a clone of recombinant cells

must find clone containing DNA of interest

probes are used

a process used to separate dna molecules

gel electrophoresis which seperates molecules based off charge size and shape

negative charged dna drawn towards + electrode

smaller fragments migrate faster then larger ones

what is agarose

makes up gel and acts as a molecular sieve in gel electrophoresis

southern blot

used to separate DNA molecules

dna is transfered from gel to nitrocellulose membrane and probes are used to localize dna sequence of interest

what is the northern plot

used to detect RNA

uses of southern blots

genetic fingerprinting

diagnosis of infectious disease

demonstrate incidence and prevalence of organisms that cannot be cultured

DNA Microarrays

consists of molecules of immobilized single stranded DNA

fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences

scientific uses of dna microarrays

monitoring gene expression

diagnosis of infection

identification of organisms in an environmental sample

the goal of dna technology

insertion of DNA into cell

natural methods of inserting dna into cells

transformation

transduction

conjugation

artificial methods of inserting dna into cells

electroporation

protoplast fusion

injection using gene gun and microinjection

genetic mapping

locating genes on a nucleic acid molecule

benefit of genetic mapping

provides useful facts concerning metabolism, growth, characteristics and relatedness to others

locating genes

until 1970 genes were identified by labor intensive methods

now they are simpler and universal methods available such as restriction fragmentation and FISH

environmental studies

scientist identify organisms through their dna fingerprints

allowed scientist to identify 500 species of bacteria from human mouths

determines that methane producing archaea are a problem in rice agriculture

pharmaceutical and therapeutic applications

protein synthesis

-creation of synthetic peptides for cloning

vaccines

genetic screening

dna microarrays used to screen individuals for inherited disease caused by mutations

can identify pathogen’s dna in blood or tissues

dna fingerprinting

identifying individuals or organisms by their unique dna sequence

gene therapy

missing or defective genes replaced with normal copies

some patients immune systems react negatively

medical diagnosis

patient specimens can be examined for presence of gene sequences unique to certain pathogens

xenotransplants

animal cells, tissues or organs introduced into human body

production of transgenic organisms (agricultural applications)

recombinant plants and animals altered by the addition of genes from other organisms

herbicide resistance

farmers can kill weeds without killing crops

salt tolerance

scientist removed gene for salt tolerance and inserted into tomato and canola plants

transgenic plants survive, produce fruit and remove salt from soil

freeze resistance

crops sprayed with genetically modified bacteria can tolerate mild freezes

pest resistence

farmers can use to reduce insect damage to crops

gene for bt toxin inserted into crop plants

agricultural applications

improvements in nutritional value and yield

BGH allows cattle to gain wright more rapdily

gene for beta carotene(vitamin a precursor) inserted into rice

supremacist view

humans are of greater value than animals

ethics and safety of recombinant dna technology

long term effects of transgenic manipulations are unknown

unforseen problems arise from new technologies and procedures

natural and genetic transfer could deliver genes from transgenic plants and animals into other organisms

transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic