Chapter 9 Study Guide

Define and Recognize:

 Clone: Population of cells arising from 1 cell; Each carry a new gene
 PCR: Polymerase Chain Reaction; Rapid process that makes billions of copies of a gene;
Makes multiple copies of piece of DNA enzymatically
 Bt Toxin: Bacillus Thuringiensis toxin; Biological insecticide; Bt toxin is produced by B.
Thuringiensis and is inserted into some plants and when insects eat it they die.
 Reverse Transcriptase: an enzyme used to generate complementary DNA (cDNA) from
an RNA template
Biotechnology and Recombinant DNA
Biotechnology: use of microorganism cells or cell components to make a products; Examples
include food, antibiotics, vitamins and enzymes
Recombinant DNA technology (rDNA): insertion or modification of genes to produce desired
proteins
Vector: self-replicating DNA used to carry desired gene to new cell; Example of a vector is a
plasmid;
Clone: population of cell arising from one cell; each carries a new gene

Figure 9.1A Modification Procedure (Study Slide)

 Take vector (like a plasmid) and recombine with DNA that has desired gene
 Put vector (plasmid) into bacteria
 Cells with genes of interest are cloned
 Purpose: To create and harvest copies of genes; create and harvest protein production
of gene
Restriction Enzymes: Study Figure 9.2

 To get into vector, they cut sequences of DNA

 Destroy bacteriophage DNA in bacterial cells
 Cannot destroy (host) Dna with methylated cytosine’s
Figure 9.3: Plasmid (vector) used for cloning

 Plasmid normally has 3 important things:

o Reaction enzyme sites
o ORI: origin of replication
o ampR: Antibiotic resistance gene
 Polymerase Chain Reaction (PCR) = routine method of DNA amplification

 Rapid process to make billions of copies of a gene; make multiple copies of a piece of a
DNA enzymatically; technique where small samples of DNA can be quickly amplified
 Used to:
o clone DNA for recombination
o amplifies DNA to detectable levels
o sequence DNA
o Diagnose genetic disease
o Detect pathogens

 Figure 9.4 PCR: 3 steps to the polymerase chain reaction

o Melt Step: Melt DNA at 94C; This breaks Dna apart and becomes single strand
DNA
o Amealing Step: At 60C, primers attach to single strand DNA
o Extension: At 72C, We have synthesized new DNA strand with Dna polymerase
that’s heat stable, called O TAQ DNA Polymerase; Builds new DNA molecule
o This process takes 1 dna strand and makes 2, 4, 6, 8, etc.

Figure 9.8 Genomic libraries (less used method to obtain DNA)

o It takes DNA from organism of interest
o Digests with reaction enzymes
o Makes thousands of clones each with different DNA fragments = “libraries”

Obtaining DNA

 Complementary DNA (cDNA): is made from mRNA by reverse transcriptase

Figure 9.9 Making cDNA for Eukaryotic Gene

 Isolate mRNA
 Make cDNA copy using reverse transcriptase ( which comes from retroviruses)
 No introns= got rid of them
Figure 9.11 Blue-white Screening (One method of selecting recombinant bacteria)

 Selection: Use of antibiotic to inhibit bacteria that don’t carry ampicillin resistance gene
on vector
 Screening: Restriction enzyme site is in LAC-Z gene; insertion of DNA of interest mutates
LAC-Z gene
  Only bacteria that picked up the plasmid will grow in the presence of ampicillin;
 X-Gal: Dye that becomes blue in the presence of LAC-Z
 So take the vector and DNA- insert in E.Coli- add to agar with ampicillin and x-gal; you
will get some blue colonies and some white colonies
o Blue Colonies: Make LAC-Z; Don’t make DNA of interest
o White Colonies: No LAC-Z; Do have DNA of interest

Making a Product (If we got DNA into vector, and all checks out)

 E. Coli (used to synthesizes gene products):

o Easily grown and known genomics
o Need to eliminate endotoxin from products
o Cells need to be lysed
 Saccharomyces cerevisiae (yeast) :
o Easily grown and known genomics
o May express eukaryotic genes easily
 Plant cells and whole plants:
o May express eukaryotic genes easily
o Plants are easily grown
 Mammalian cells:
o May express eukaryotic cells easily
o Harder to grow

Therapeutic Applications (How we use it)

 Human enzymes and other proteins

 Subunit vaccines
 Nonpathogenic viruses carrying genes for pathogen’s antigens as DNA vaccines
 Gene Therapy to replace defective or missing genes
 Example of application is Hep B Vaccine
Scientific Applications

 Understanding DNA
 Sequencing organisms’ genomes
 DNA fingerprinting for identification (track infectious diseases and where the outbreak
originated)
Agrobacterium: Engineering bacteria for agriculture; well known for its ability to transfer DNA
between itself and plants;
Some benefits include:
 Bt toxin
 Herbicide resistance
 Suppression of genes: Antisense DNA
 Nutrition
 Human Proteins