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“Tools and Methods of Genetic Engineering” Ch 20
Restriction Enzymes fig 20.3
1. “molecular scissors”
2. cut DNA at specific palidromic sequences
3. creates sticky ends (which allows cutting and pasting different DNAs)
4. sticky ends sealed with ligase  permanent recombinant DNA
Replica Plating fig 20.4
1. using felt to transfer colonies from old petri dish (“master plate”) to new one, thus
making a replication/copy of the old colonies
2. master plate is not used for experimenting, replicas are used instead
Nucleic acid hybridization fig 20.5
1. using a short-single stranded DNA or RNA for detecting the presence of a particular
gene in your unknown DNA
2. uses autoradiography to visualize the presence of the probe (radioactive probe
develops the photographic film)
3. used for
a. finding recombinants among non-recombinants
b. identifying common sequences among related species (ex. Fly & human
cancer genes)
cDNA needed for bacteria expression (NO SPLICEOSOMES IN BACTERIA)
1. DNA created from RNA using reverse transcriptase
2. bacteria do not have splicesomes, therefore, human cDNA must be used because
regular human DNA has introns and can not be converted to proteins in bacteria
genomic library pg 389
1. human DNA is too large for 1 bacteria to carry
2. human DNA is cut (with restriction enzymes) into thousand of short fragments and
then each of those short fragments is inserted to separate bacteria
3. a “complete” copy of human genome has been accomplished in 2002 = human
genomic library (human genome project)
4. problems: expensive to maintain, gene may be cut into several pieces
Polymerase chain reaction (PCR) pg 391
1. method for making many copies of a specific segment of DNA
2. used in crime scene analysis when only tiny samples are present (ie. Drop of blood, a
few hairs, skin under fingernails, etc)
3. alternates heat (denaturation) and cooling (RNA primers & DNA polmerase) by about
20 times to create enough DNA to perform studies
4. A single copy of DNA is not enough to do analyses upon, so many copies are created
Gel electrophoresis fig 20.8, page 393
1. separates macromolecules (DNA, RNA, proteins) on the basis of their rate of
movement through a gel under the influence of an electric field
2. separates molecules on their size (length of DNA, etc) and charge (functional groups,
etc.)
3. RFLPs (restriction fragment length polymorphisms) = “zebra code” of molecules
4. crime scene analysis, everyone (except identical twins) have unique RFLPs
Fluorescence In situ Hybridization “FISH” (chromosome location of gene) fig 20.11
1. using nucleic acid probe to find the general location of the gene on the chromosome
2. locating gene in different species
Human Genome Project pg 398
1. map the entire human genome (entire DNA sequence)
2. yeast, nematode, fruit fly, mouse = completely sequenced
3. 3 steps:
a. genetic (linkage) mapping
b. physical mapping = ordering DNA fragments = chromosome walking fig 20.8
c. DNA sequencing = Sanger method pg 397
Genomic analysis pg 400
1. analyzing DNA sequences = study good vs poor promoters, exon shuffling = create
new genes, evolutionary relationships
2. studying gene expression = how are genes timed for expression?, DNA microarray
assays fig 20.14 pg 401 = compare cancerous tissues with noncancerous,
discovering photosynthetic enzymes by comparing roots with leaves
3. determing gene function = in vitro mutagenesis = alter one protein and see what
happens to the cell
4. genomics = study of genomes and gene based on DNA sequences
Practical applications of DNA technology
1. Medicine
a. diagnosis of diseases
b. human gene therapy fig 20.16
c. pharmaceutical products = human growth hormone, tissue plasminogen
activator
d. antisense nucleic acids = block bad mRNAs
e. vaccines
2. Forensics = DNA fingerprinting fig 20.17
3. Environmental = bacteria that can digest oil, extract harmful metals, etc
4. Agricultural
a. Transgenic animals
b. Plants = Ti plasmid fig. 20.19
1. Nitrogen-fixation problem