restriction endonucleases are...

2 big uses for these

enzymes that cleave DNA at specific sites

Allow physical mapping

allow creation of recombinant DNA

2 types of restriction enzymes:

Type 1:

Type II:

Type 1: Cut near restriction site

Type II: Cut at the restriction sites

(sites are palindromes)

Gel Electrophoresis

technique used to seperate DNA fragments by ____

agorose or polyacrylamide gel is subjected to ____ __

DNA moves toward ____ pole

DNA is visualized using ___ ___

size

electric field

positive

fluorescent dyes

the isolation of a specific DNA sequence (usually protein-encoding)

sometimes called gene cloning

plasmids are...

3 components?

small, circlar extrachromosomal DNA molecules that can self replicate

1. origin of replication
2. selectable marker
3. multiple cloning site (MCS)

phage vectors are modified ____ ____

based on lambda phage of E. Coli

used to clone inserts of up to ____ kbp

have 2 features not shared w/ plasmid vectors:

bacterial viruses

40

1. kill host cells
2. linear genomes (middle replaced w/ inserted DNA)

artificial chromosomes are used to ...

BAC stands for?

YAC?

 clone very large DNA fragments

bacterial artificial chromosomes

yeast artificial chromosomes

only the expressed part of the genome

(complimentary DNA) - synthesized from mRNA using revers transcriptase

molecular hybridization

Annealing???

is a technique used to identify specific DNAs in complex mixtures

(DNA/RNA probes)

1. plating the library
2. replicating the library
3. screening the library

southern blotting

• A sample ____ is digested by restriction enzymes and separated by gel electrophoresis

• gel is transferred onto a ____ ___

• then ____ with a cloned, radioactively-labeled DNA probe
• complementary sequences are revealed by ______

DNA

nitrocellulose filter

hybridized

autoradiography

western blotting

_____ are electrophoresed and then blotted onto the filter

detection requires an _____ that can bind to one protein

proteins

antibody

RFLP analysis

restriction fragment length polymorphisms (RFLPs) are generated by...

these fragments are often not _____ in different individuals

can be detected by ___ blotting

 point mutations or sequence duplications

identical

southern

DNA fingerprinting

an identification technique used to detect differences in the DNA of individuals

DNA sequencing

a set of known _____ is generated

separated by high resolution ___ ____ resulting in a ladder

sequence is read from (top down/ bottom up)

fragments

gel electrophoresis

bottom up

DNA sequencing : enzymatic technique is powerful, but it is ___ ___ and ___ - ____

automated techniques are faster and practical:

___ ___ are used instead of radioactive labels

reaction is done in how many tubes?

data are assembled by a ____

labor intensive and time-consuming

fluorescent dyes

one tube

computer

Polymerase chain reaction (PCR)

3 steps?

allows the amplification of small DNA fragment using primers that flank the region

1. denaturation - high temp
2. annealing of primers - low temp
3. DNA synthesis - intermediate temp, taq polymerase

forensics

detection of genetic defects in embryos

analysis of mitochondrial DNA from early human species

in vitro mutagenesis

ability to create mutations at any site in a ___ gene

has been used to produce ___ ___, in which a known gene is inactivated

cloned

knockout mice

Medically important proteins for humans can be produced in ____

examples?

bacteria

human insulin

interferon

atrial peptides

tissue plasminogen activator

human growth hormone

Gene therapy

adding a functional copy of a gene to correct a __ __

what is an example? good and bad about it?

hereditary disorder

severe combined immunodeficiency disease (SCID)

succesful at first, causes rare leukemia later

Ti (tumor inducing) plasmid is the most used vector for plant ___ ___

normally infects __ ___

bacterium doesn't infect __, ___ and ___

genetic engineering

broadleaf plants

corn, rice, and wheat

gene guns

uses bombardment of tiny  _ __ coated with DNA

possible for any species, however the copy number of ___ ___ cannot be controlled

gold particles

inserted genes

herbicide resistance

broadleaf plants have been engineered to be resistant to the herbicide ____

this allows for no-till planting

glyphosate

pest resistance

___ proteins have been tranferred into crop plants to make them pest resistant

__ ___ from bacillus thuringiensis

insecticidal

Bt toxin

Golden rice

rice that has been genetically modified to produce ____ ____, which is converted in the body to vitamin A

crop safety

movement of genes into wild relatives

loss of biodiversity

biopharming

transgenic plants are used to produce phharmaceuticals like:

human serum albumin

recombinant subunit vaccines

recombinant monoclonal antibodies

transgenic animal technology has not been as succesful aas that in ___

one example of transgenic animal tech.

plants

enviropig