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AP Biology
Biotechnology
and
Genetic Engineering
AP Biology
Biotechnology Today
Genetic Engineering: process of manipulating genes and
genomes
Biotechnology: process of manipulating organisms or their
components for the purpose of making useful products.
Recombinant DNA: DNA that has been artificially made, using
DNA from different sources
eg. Human gene inserted into E.coli
Gene cloning: process by which scientists can product
multiple copies of specific segments of DNA that they can
then work with in the lab
AP Biology
Techniques of Genetic Engineering
Transformation: bacteria takes up plasmid (w/gene of interest)
Gel electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current (DNA migrates to + pole)
PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells
DNA microarray assays: study many genes at same time
AP Biology
Bacteria
Bacteria review
• one-celled prokaryotes
• reproduce by mitosis
• binary fission
• rapid growth
• generation every ~20 minutes
• 108 (100 million) colony overnight!
• dominant form of life on Earth
• incredibly diverse
AP Biology
Bacterial Genome
• Single circular chromosome
• haploid
• naked DNA
• no histone proteins
• ~4 million base pairs
• ~4300 genes
• 1/1000 DNA in eukaryote
AP Biology
Transformation
Bacteria are opportunists
• pick up naked foreign DNA
wherever it may be hanging out
• have surface transport proteins that are
specialized for the uptake of naked DNA
• import bits of chromosomes from
other bacteria
• incorporate the DNA bits into their
own chromosome
• express new genes
• transformation
(form of recombination)
mix heat-killed pathogenic & non-pathogenic bacteria
mice die
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AP Biology
Plasmids
Small supplemental circles of DNA 5000 - 20,000 base pairs
self-replicating
• carry extra genes
2-30 genes
genes for antibiotic resistance
• can be exchanged between bacteria
bacterial sex!!
rapid evolution
• can be imported from environment
AP Biology
Plasmids
• A way to get genes into bacteria easily
• insert new gene into plasmid
• insert plasmid into bacteria = vector
• bacteria now expresses new gene
• bacteria make new protein
+
transformed
bacteria gene from
other organism
plasmid
cut DNA
recombinant
plasmid
vector
glue DNA
AP Biology
Biotechnology
Plasmids used to insert new genes into bacteria
gene we want
cut DNA
cut plasmid DNA
insert “gene we want” into plasmid...
“glue” together
ligase
like what? …insulin …HGH …lactase
recombinant plasmid AP Biology
Restriction Enzymes (RE)
• restriction endonucleases
• discovered in 1960s
• evolved in bacteria to cut up foreign DNA
“restrict” the action of the attacking organism
protection against viruses & other bacteria
AP Biology
Restriction Enzymes (RE)
radar
racecar
Madam I’m Adam
Able was I ere I saw Elba
a man, a plan, a canal, Panama
Was it a bar or a bat I saw?
go hang a salami I’m a lasagna hog
palindromes
AP Biology
Restriction enzymes (RE)
• Action of enzyme
• cut DNA at specific sequences
restriction site
• symmetrical “palindrome”
• produces protruding ends
sticky ends
• will bind to any complementary DNA
• Many different enzymes
• named after organism they are found in
EcoRI, HindIII, BamHI, SmaI
restriction enzyme animation
CTGAATTCCG
GACTTAAGGC
CTG|AATTCCG
GACTTAA|GGC
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AP Biology
Restriction enzymes (RE)
Cut DNA at specific sites
leave “sticky ends”
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA
GTAACGAATTCACGC
TT
CATTGCTTAAGTGCG
AA
restriction enzyme cut site
restriction enzyme cut site
AP Biology
Sticky Ends
Cut other DNA with same enzymes
leave “sticky ends” on both
can glue DNA together at “sticky ends”
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA gene
you want
GGACCTG AATTCCGGATA
CCTGGACTTAA GGCCTAT
chromosome want to add
gene to
GGACCTG AATTCACGCTT
CCTGGACTTAA GTGCGAA combined
DNA
AP Biology
Sticky Ends
TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCA AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGT
gene you want cut sites cut sites
isolated gene sticky ends
chromosome with new gene added
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACG CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATG
sticky ends together DNA ligase joins the strands Recombinant DNA molecule
GAATTCTACGAATGGTTACATC AGATGCTTACCAATGTAGCGG
TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTC AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAG
AP Biology
Making Recombinant DNA (rDNA)
Restriction enzymes
(RE)
cut gene of interest
and
DNA ligase pastes it into
new plasmid
AP Biology
Gene Cloning 1. CUT
Restriction enzymes cut DNA into fragments that can be isolated and separated
- very specific proteins, recognizes and cuts DNA at specific sequence into pieces
Ex: EcoRI – cuts DNA whenever C-T-T-A-A-G sequence occurs
2. PASTE Production of recombinant DNA (rDNA)
- DNA composed of fragments of DNA
segments from at least two different organisms
- restriction enzymes cut bacterial plasmids (extra circular DNA molecules in bacteria)
- plasmids have “sticky ends” (unpaired bases)
- original DNA is attached to plasmid sticky ends
- ligase stitches rDNA together AP Biology
Gene Cloning
3. INSERT & COPY Reintroduction of DNA into bacterial (cloning) vector
- recombinant DNA taken up with bacterial DNA and now produced by bacterial cell
- recombinant DNA is isolated and CLONED
- PCR (polymerase chain reaction) induced
- purpose: to make 1000s of recombinant plasmids PCR animation
4. FIND DNA sequencing
- Process of reading exact order of bases in fragment of DNA
- makes it possible for scientists to make sure gene of interest has been cloned gene cloning video
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AP Biology
Gene Cloning (short version)
grow
bacteria
harvest (purify)
protein
transformed
bacteria
plasmid
gene from
other organism
+
recombinant
plasmid
vector
AP Biology 2005-
2006
Applications of Gene Cloning
AP Biology
Applications of Cloning: Genomic Libraries
Cut up all of nuclear DNA
from many cells of an
organism
restriction enzyme
Clone all fragments into
many plasmids at same
time
“shotgun” cloning
Create a stored collection
of DNA fragments
petri dish has a
collection
of all DNA
fragments from the
organism
AP Biology
Applications of Cloning:
cDNA(complementary DNA) Libraries
Collection of only the coding sequences of expressed genes
• extract mRNA from cells
• reverse transcriptase
RNA DNA
from retroviruses
• clone into plasmid
Applications
need edited DNA for expression in bacteria
Ex: human insulin
animation
AP Biology
Applications of Cloning: Entire Organisms
Nuclear transplantation: nucleus of egg is removed
and replaced with nucleus of body cell
AP Biology
Problems with Reproductive Cloning
• Cloned embryos exhibited various defects
• DNA of fully differentiated cells have epigenetic changes
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AP Biology
Applications of Cloning: Stem Cells
Stem cells
unspecialized cells- can
reproduce itself indefinitely and
differentiate into other specialized
cells
Types:
- Zygote stem cells:
totipotent (any type of cell)
- Embryonic stem cells:
pluripotent (many cell types)
- Adult stem cells (somatic):
- multipotent (a few cell types)
- induced pluripotent
(re- programmed)
AP Biology
Stem Cells for Disease Treatment
AP Biology
Gel Electrophoresis
A method of separating DNA
fragments of different size in a
gelatin-like material using an
electrical field
DNA is negatively charged
when it’s in an electrical field it
moves toward the positive side
+ –
DNA
“swimming through Jello” AP Biology
DNA moves in an electrical field…
so how does that help you compare DNA
fragments?
size of DNA fragment affects how far it travels
small pieces travel farther
large pieces travel slower & lag behind
Gel Electrophoresis
+ –
DNA
“swimming through Jello”
AP Biology
Gel Electrophoresis
longer fragments
shorter fragments
power source
completed gel
gel
DNA & restriction enzyme
wells
-
+ AP Biology
Southern Blotting
Once banding patterns are apparent they are
transferred to a Southern Blot for identification
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AP Biology
DNA Fingerprints
Comparing blood samples on
defendant’s clothing to
determine if it belongs to
victim
DNA fingerprinting
comparing DNA banding
pattern between different
individuals
~unique patterns
Bozeman video
Naked science video
AP Biology
Uses: DNA Fingerprinting in Forensics
Comparing DNA sample from crime scene with
suspects & victim
–
+
S1
DNA
S2 S3 V
suspects crime scene sample
AP Biology
RFLPs Restriction Fragment Length Polymorphism
• differences in DNA between individuals
• due to differences in “junk” DNA
change in DNA sequence affects
restriction enzyme “cut” site
creates different fragment sizes &
different band pattern
Restriction fragment length polymorphisms
AP Biology
RFLPs in Forensics
AP Biology
Uses: Evolutionary Relationships
Comparing DNA samples from different organisms to measure evolutionary relationships
–
+
DNA
1 3 2 4 5 1 2 3 4 5
turtle snake rat squirrel fruitfly
AP Biology
Uses: Disease Diagnosis
Comparing normal allele to disease allele
chromosome with
disease-causing
allele 2
chromosome
with normal
allele 1 –
+
DNA
Example: test for Huntington’s disease
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AP Biology
Uses: Paternity Tests
AP Biology
Polymerase Chain Reaction (PCR)
Method for amplifying (making many copies) of a specific segment of DNA
Materials needed:
• template strand from DNA of one cell
• DNA polymerase enzyme
• Nucleotides
• ATP, GTP, CTP, TTP
• Primer (define section
of DNA to be cloned)
AP Biology
Microarray “DNA Chips”
Slide with a sample of
each gene from the
organism
•each spot is one
gene
animation
AP Biology
Applications of Genetic Engineering Gene Therapy Using a Retroviral Vector
AP Biology
Applications of Genetic Engineering
“Pharm” animal: produce human protein secreted in milk for medical use
AP Biology
Applications of Genetic Engineering
STR Analysis (Short Tandem Repeats)
Non-coding DNA has regions with sequences (2-5 base length)
that are repeated
Each person has different # of repeats at different locations
(loci)
Current method of DNA fingerprinting used – only need 20
cells for analysis
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AP Biology AP Biology
Applications of Genetic Engineering
Medicines and Vaccines
produced by bacteria and
viruses
- E coli: used to make human insulin
- hamster cell cultures: used to make TPA (tissue plasminogen activator)- dissolves blood clots in heart attacks
- EPO (erythropoiten): increase red blood cell production
- interferon: fights viral infections, increases immunity
- vaccines
vaccine animation
AP Biology
Applications of Genetic Engineering Pollution Control
Pollution control
- genetically altered
bacteria used to
decompose garbage
sewage, and
petroleum products
Unlike the left tower, which uses
chemicals, the tower on the right at this
wastewater-treatment plant now uses
bacteria-covered foam blocks (inset) to
eliminate the hydrogen sulfide bubbling
from treated sewage
AP Biology
Applications of Genetic Engineering
Transgenic Organisms
Genetically Modified Organisms (GMOs)
• organisms that contain foreign genes
Transgenesis
The use of recombinant DNA techniques to
introduce new characters (ie. genes) into
organisms (including humans) that were not
present previously.
AP Biology
Applications of Genetic Engineering
GMOs
enabling plants to produce new proteins
Protect crops from insects: BT corn
corn produces a bacterial toxin that kills
corn borer (caterpillar pest of corn)
Extend growing season: fishberries
strawberries with an anti-freezing gene
from flounder
Improve quality of food: golden rice
rice producing vitamin A
improves nutritional value
AP Biology
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AP Biology AP Biology
Human Genome Project
• U.S government project (1990) – 15 year
timeframe
• goal was to sequence entire human
genome
3 billion base pairs
• Completed in 2003
• Blueprint of a human
• Genomes sequenced thus far*: 58,000 prokaryotes, 2700 eukaryotes, 5300 viruses
AP Biology
Comparison of Genomes
Organism Genome Estimated
Size Genes
Human (Homo sapiens) 3 billion 30,000
Laboratory mouse (M. musculus) 2.6 billion 30,000
Mustard weed (A. thaliana) 100 million 25,000
Roundworm (C. elegans) 97 million 19,000
Fruit fly (D. melanogaster) 137 million 13,000
Yeast (S. cerevisiae) 12.1 million 6,000
Bacterium (E. coli) 4.6 million 3,200
Human Immunodeficiency Virus (HIV) 9700 9
AP Biology
Human DNA
• 3 billion base pairs
• ~20,000 genes
• Only 1.5% codes for
proteins (or RNA)
• Mostly Repetitive DNA:
sequences present in
multiple copies
SNP video
AP Biology
Evolution of Genomes
Interspersed Repetitive DNA
Repetitive DNA is spread throughout genome
• interspersed repetitive DNA (SINEs- Short
INterspersedElements) make up 25-40% of mammalian
genome
• in humans, at least 5% of genome is made of a family of
similar sequences
• 300 bases long
• Aluis an example of a "jumping gene" called a transposon;
AP Biology
Evolution of Genomes
Transposable Elements (jumping genes)
- Make up 75% of repetitive DNA
- DNA sequence that "reproduces" by copying itself &
inserting into new chromosome locations
- Can be moved from one location to another in genome
- Discovered by Barbara McClintock – corn breeding experiments
• 2 Types:
Transposons
Retrotransposons
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AP Biology
Evolution of Genomes Rearrangements in the Genome
Transposons
•Move within genome via DNA
intermediate
•Insertion of transposon sequence in
new position in genome
•“cut & paste” or “copy & paste” mechanisms
•Requires enzyme transposase
Insertion sequences cause
mutations when they happen to land
within the coding sequence of a
gene or within a DNA region that regulates gene expression
AP Biology
Evolution of Genomes
Retrotransposons
• Most transposons are retrotransposons
• Move within a genome via RNA intermediate,
transcript of the retrotransposon DNA.
• Involves enzyme reverse transcriptase
AP Biology
Evolution of Genomes
Insertion effects of transposons:
Can interrupt or alter gene function
Multiple copies of genes
Duplication genes with related functions
Genes diverge by accumulating mutations
Some become nonfunctional pseudogenes
Eventually, new genes with new functions can occur
AP Biology
Evolution of Genomes
Multigene Families
• Collections of 2 or more identical or very similar genes
• Eg. hemoglobin: -globin and -globin gene families
AP Biology
Evolution of Genomes
Transpositions chromosomal rearrangements
AP Biology
Evolution of Genomes
Transposable elements contribute to evolution
• Promote recombination, disrupt genes or control elements, & carry genes to new locations
• May be harmful or lethal, but can also have small beneficial effects
• Provides raw material for natural selection
• Ex: antifreeze genes in fish (antifreeze proteins- AFP)
• Aids survival in sub zero environments (binds to ice crystals)
• Old protein gene mutates duplicates divergent evolution
HHMI video