Chapter 17 Flashcards
What is biotechnology?
use of biological agents to make technological advances
Applied biotechnology
- medicine
vaccine development, drug discovery - agriculture
GMOs and crop yields - energy
biofuels, environmental remediation
isolation and extraction of DNA from cells procedure
- break cell wall
lysis buffer with SDS - Degrade macros
proteases and ribonucleases - remove and charge proteins
“salting out” with NaCl - precipitate and remove contaminants
wash with ethanol - rinse and remove target DNA
elution buffer with tris-EDTA - storage at -80 C
Gel Electrophoresis
Separation and visualization technique
gel creates “pores”
molecules separate based on size
gel between two electrodes and submerged in buffer
Gel used for electrophoresis in proteins
acrylamide gel (verticle)
Gel used for electrophoresis in DNA
agarose gel (horizontal)
How do solutions separate in gel electrophoresis
mass/charge ratio
fragments have negative charge so move toward positive end
smaller fragments move farther
negative electrode gel electrophoresis
Cathode
positive electrode gel electrophoresis
Anode
Dyes used in electrophoresis
“walk up DNA stairs”
ethidium bromide
super green (less toxic)
Southern blotting techniques
DNA
Northern blotting techniques
RNA
Western blotting techniques
Protein
Polymerase Chain Reaction
used for rapid DNA replication
billions of copies within hours
now fully automated
Polymerase Chain Reaction steps
- Denaturation
high heat 95 C
double -> single strands - Annealing
50 to 60 C
primer binds to strands - Extension
72 C
Taq DNA polymerase
adds complementary bases - each cycle doubles DNA
How many cycles are needed in PCR
20-30
Problems with PCR
No proofreading
mistakes in copying possible
DNA cloning
isolation of DNA carrying a gene, amplify DNA, produce multiple copies of one gene
Why is identifying genes on chromosomes diffficult
100s of genes/chromosome
each gene may be 1/100,000 of DNA long
Bacterial Plasmids
Accessory “circular” DNA not needed for normal function is easily inverted to sequence of interest
cloning vectors carry DNA into host cell and are incorporated into genome
instances of reproductive cloning
mammals specifically
1997: Dolly the sheep
cloned successfully from adult cells which resulted in early arthritis, euthanized at age 6
2018: primates (not USA)
Therapeutic cloning
nose/ear grown
pig heart transplant
produce various cell types/provide cells/tissue for treatment.
Practical applications: disease/diagnostic treatment
PCR to confirm pathogens (COVID 19)
higher specificity than protein assay
less likely to receive false negative
Practical applications: Personalized genomic analyses
personalized medicine
genetic profile generated
predict treatment effectiveness
decide on treatment regime
especially for breast cancer (120 types)
Practical applications: Gene therapy
introduction of genes via virus to train immune system
specifically for afflicted individuals
with the intent to cure disorder
cloned genes modify a human
Transgenic organisms
include recombinant DNA
mendel’s pea breeds
drought resistant corn
bacterial transformation
Foreign DNA/genes inserted
pesticide resistance in crops
luminescence proteins in fish
growth hormone in salmon
Ethical Considerations for GMOs
could create new/unintended pathogens
genetically cripples/cannot survive outside lab
pass genes to natural relatives
produce toxic proteins
labelling dilemma
The Human Genome Project
NIH and DOE0ORNL
ID 20-25k human genes
sequence 3 billion bases
92% DNA (exons) OCT 1990-APR 2003
also E. Coli, fruit fly, lab mouse
Telomere to Telomere (T2T) consortium
national human genome research institute
why sequence the human genome?
analyze genes for genetic mutations/diseases
understand effects of genetic variation
study human evolution
develop personalized medicine
Steps to DNA sequencing
- Nucleic acid hybridization
base pairing to complementary strand - Determine nucleotide order
specific gene sequence
Sanger sequencing
Frederick Sanger NP 1980
“Dideoxy sequencing” genes of interest
dideoxynucleoside triphosphates anneal, fluorescent markers for each base contained
Adenine fluorescent marker
green
Thymine fluorescent marker
red
Guanine fluorescent marker
black
Cytosine fluorescent marker
blue
Next generation of DNA sequencing
“high throughout”
Identify and amplify sequence of interest
sequencing by synthesis
computer reads nucleotides
complements added real time
Current lab sequencers (availability)
over time has become much more affordable and efficient
Nanopore sequencers
no denature/amp
entire DNA molecule at once
DNA passed through protein pore and each nucleotide is read as a change in electrical signal as it goes through
Restriction enzymes
anti bacteriophage (virus for bacteria) mechanism restricts viral growth
IDs and removes foreign DNA
enzymes target specific restriction site
Methylation
protects self-DNA in bacteria against restriction enzymes
Restriction enzymes process
- R enzyme cut at restriction site
- plasmid and sequence of interest are cut
- insert sequence between fragments
- base pairing at new “sticky ends”
- ligase seals new DNA-
Clustered regularly interspersed short palindromic repeats (CRISPR)
adapted from naturally occurring gene editing sys in bacteria (pro)
target specific sequences for removal or replacement
endonuclease enzyme in CRISPR
- Cas 9 (bacterial anti-virus) cuts DNA/inactivates viruses
- specififc nucleotide guide sequence PAM (protospacer adjacent motif) has cut made adjacent
cut made inside DNA
CRISPR system
- target specific sequence in organism
nuclease causes break in DNA - Knock OUT or IN
- Compare to wildtype
Knock OUT CRISPR
mutate the gene -> inactivates
Knock IN CRISPR
insert new sequence into gene
DNA fingerprinting
small amount of DNA needed, PCR amplifies
identifies person based on DNA
old DNA fingerprinting method
entire genome -> restriction enzymes
different restriction sites -> DNA fragments
use gel electrophoresis to create bands
short tandem repeat profiling
modern DNA fingerprinting method
no restriction enzymes, short sequences (GATA)
recuring DNA
PCR amplifies targeted DNA
labeled with dye
DNA sequence
Detector records length
FBI cultivates large ____ database
STR
short tandem repeat profiling
Pharmacogenomics
drug effectiveness
linked to gene sequence
Metagenomics
study of collective genomes
multiple species interactions
Proteomics
entire set of proteins by cell
study of proteome
Metabolomics
all small metabolites in organism
based on genetic makeup
study metabolomes
Which way does DNA flow in gel electrophoresis
Cathode to anode
negative to positive
Why is Taq DNA polymerase used in PCR
because it doesn’t denature at 72 C
