MicroLecE2Ch9

Question 1

Biotechnology

Answer 1

Use of microorganisms, cells, or cell components to make a product
Exs: foods, antibiotics, vitamins, enzymes

Question 2

Recombinant DNA (rDNA) technology

Answer 2

Insertion or modification of genes to produce desired proteins

Question 3

Vector

Answer 3

Self-replicating DNA used to carry the desired gene to a new cell

Question 4

Plasmid

Answer 4

Most common type of vector
Small circular piece of DNA
Non-essential
Ex F-factor used in conjugation

Question 5

Clone

Answer 5

Population of cells arising from one cell, each carries the new gene, all identical

Question 6

Genetic Modification Procedure

Answer 6

Vector is isolated
DNA is cut by a restriction enzyme into fragments resulting in DNA containing gene of interest
Gene is inserted into plasmid
Plasmid taken up by a cell such as a bacterium which transforms the bacterium
Cells w/gene of interest are cloned
Goal is to either make copies of the gene (pest resistance, cleaning up toxic waste) OR to make protein product of the gene (human growth hormone treats stunted growth)

Question 7

Selection

Answer 7

Allows for selecting which microbe has the desired product

Question 8

Mutation

Answer 8

Mutagens cause mutations that might result in a microbe w/a desirable trait
Ex: some vaccines

Question 9

Site-Directed Mutagenesis

Answer 9

Change a specific DNA code to change a protein

Question 10

Restriction Enzyme/Restriction Endonuclease

Answer 10

First step of cloning is cutting open the plasmid
Each restriction enzyme cuts a specific sequence of DNA
Discovered in bacteria which naturally produce these enzymes as a defense mechanism
Destroys bacteriophage DNA in bacterial cells, protects its own DNA w/methyl groups on the surface
Every restriction enzyme site is specific and a palindrome (reads same forward and backward)

Question 11

Polymerase Chain Reaction (PCR)

Answer 11

Can make millions of copies of DNA from a small amount of DNA
Requires target DNA, RNA Primer, DNA Polymerase, nucleotides A/T/C/G, ATP
1) First Cycle:
Melting: incubate target DNA at 90 C to separate the strands by melting the hydrogen bonds
Primer Binding: primers attach to single-stranded DNA during incubation at 60 C
Extension: incubate at 70 C, during this time two copies of target DNA are formed
2) Second Cycle:
Repeats the first cycle

Question 12

Inserting Foreign DNA Into Cells

Answer 12

5 ways –

1) Electroporation: most common. Electroshock the bacteria allowing it to expand and the pores to open
2) Transformation: uptake of naked DNA. Uses a chemical to open the pores
3) Protoplast Fusion
4) Gene gun
5) Microinjection

Question 13

Genomic Libraries

Answer 13

Made of pieces of an entire genome stored in plasmids, phages, or yeast cells

Question 14

Complementary DNA (cDNA)

Answer 14

Eukaryote
Makes DNA copy of the mature mRNA that already has intron removed
Then DNA can be stored and active genes are ready for use

Question 15

Selecting a Clone

Answer 15

1) Plasmid DNA and foreign DNA both cut w/same restriction enzyme. Plasmid has genes for lacZ and ampicillin resistance
2) Foreign DNA will insert into the lacZ gene. Bacterium receiving the plasmid vector will not produce X-gal if foreign DNA has been inserted into the plasmid
3) Recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant
4) All treated bacteria put on plate containing ampicillin and lacZ
5) Only bacteria that picked up the plasmid will grow in presence of ampicillin. Bacteria that hydrolyze X-gal produce blue colonies. Bacteria that cannot hydrolyze X-gal produce white colonies which means it has the actual gene inserted into the plasmid

Question 16

Making a Product

Answer 16

1) E. coli: easily grown, genomics are known, need to eliminate endotoxin from products, cells must be lysed to get product
2) S. cerevisiase: easily grown, genomics known, may express eukaryotic genes easily
3) Plant cells and whole plants: easily grown, may express eukaryotic genes easily
4) Mammalian cells: harder to grow, may express eukaryotic genes easily

Question 17

Therapeutic Applications

Answer 17

1) Human enzymes and other proteins
2) Subunit vaccines
3) Nonpathogenic viruses carrying genes for pathogen’s antigens as DNA vaccines
4) Gene therapy to replace missing or defective genes

Question 18

RNA Interference (RNAi)

Answer 18

Occurs in cytoplasm
Used to silence a gene causing problems
Interfered w/ability of the mRNA to bind to the ribosome

Question 19

Random Shotgun Sequencing

Answer 19

Tells the nucleotide sequence but not where gene is located or fxn
1) Constructing a gene library:
isolate DNA
cut DNA w/restriction enzymes
clone DNA in a BAC
2) Random Sequencing: sequence DNA fragments
3) Closure phase: 
assemble sequences
edit sequences, fill in gaps

Question 20

Human Genome Project

Answer 20

Goal was to discover the nucleotide sequence

Question 21

DNA Electrophoresis

Answer 21

Agarose gel - square of agar
1) put holes in agar aka “wells” towards the top and put DNA in those wells
2) run electric charge through the gel and the DNA will migrate through the gel towards the positive end/bottom of the gel b/c DNA is negatively charged
3) shorter pieces of DNA will migrate faster and be closer to bottom end of the gel, longer pieces of DNA will migrate slower and be closer to the well
EcoR1
PCR used more now than DNA Electrophoresis b/c faster and more accurate

Question 22

Southern Blotting

Answer 22

Same as DNA Electrophoresis except at the end the filter is exposed to a radioactively labeled probe for a specific gene. Probe will base-pair w/a short sequence on the gene
Filter is exposed to X-ray film. Fragment containing the gene of interest is ID’d by a band on the developed film

Question 23

Agrobacterium

Answer 23

How plants are genetically modified. Produces a plant that has the agrobacterium in it