Bio 111 > Chapter 17 > Flashcards

Chapter 17 Flashcards

(58 cards)

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1
Q

What is biotechnology?

A

use of biological agents to make technological advances

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2
Q

Applied biotechnology

A
  1. medicine
    vaccine development, drug discovery
  2. agriculture
    GMOs and crop yields
  3. energy
    biofuels, environmental remediation
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3
Q

isolation and extraction of DNA from cells procedure

A
  1. break cell wall
    lysis buffer with SDS
  2. Degrade macros
    proteases and ribonucleases
  3. remove and charge proteins
    “salting out” with NaCl
  4. precipitate and remove contaminants
    wash with ethanol
  5. rinse and remove target DNA
    elution buffer with tris-EDTA
  6. storage at -80 C
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4
Q

Gel Electrophoresis

A

Separation and visualization technique
gel creates “pores”
molecules separate based on size
gel between two electrodes and submerged in buffer

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5
Q

Gel used for electrophoresis in proteins

A

acrylamide gel (verticle)

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6
Q

Gel used for electrophoresis in DNA

A

agarose gel (horizontal)

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7
Q

How do solutions separate in gel electrophoresis

A

mass/charge ratio
fragments have negative charge so move toward positive end
smaller fragments move farther

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8
Q

negative electrode gel electrophoresis

A

Cathode

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9
Q

positive electrode gel electrophoresis

A

Anode

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10
Q

Dyes used in electrophoresis

A

“walk up DNA stairs”
ethidium bromide
super green (less toxic)

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11
Q

Southern blotting techniques

A

DNA

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12
Q

Northern blotting techniques

A

RNA

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13
Q

Western blotting techniques

A

Protein

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14
Q

Polymerase Chain Reaction

A

used for rapid DNA replication
billions of copies within hours
now fully automated

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15
Q

Polymerase Chain Reaction steps

A
  1. Denaturation
    high heat 95 C
    double -> single strands
  2. Annealing
    50 to 60 C
    primer binds to strands
  3. Extension
    72 C
    Taq DNA polymerase
    adds complementary bases
  4. each cycle doubles DNA
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16
Q

How many cycles are needed in PCR

A

20-30

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17
Q

Problems with PCR

A

No proofreading
mistakes in copying possible

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18
Q

DNA cloning

A

isolation of DNA carrying a gene, amplify DNA, produce multiple copies of one gene

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19
Q

Why is identifying genes on chromosomes diffficult

A

100s of genes/chromosome
each gene may be 1/100,000 of DNA long

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20
Q

Bacterial Plasmids

A

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

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21
Q

instances of reproductive cloning

A

mammals specifically
1997: Dolly the sheep
cloned successfully from adult cells which resulted in early arthritis, euthanized at age 6
2018: primates (not USA)

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22
Q

Therapeutic cloning

A

nose/ear grown
pig heart transplant
produce various cell types/provide cells/tissue for treatment.

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23
Q

Practical applications: disease/diagnostic treatment

A

PCR to confirm pathogens (COVID 19)
higher specificity than protein assay
less likely to receive false negative

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24
Q

Practical applications: Personalized genomic analyses

Study These Flashcards
A

personalized medicine
genetic profile generated
predict treatment effectiveness
decide on treatment regime
especially for breast cancer (120 types)

25
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
26
Transgenic organisms
include recombinant DNA mendel's pea breeds drought resistant corn bacterial transformation
27
Foreign DNA/genes inserted
pesticide resistance in crops luminescence proteins in fish growth hormone in salmon
28
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
29
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
30
Telomere to Telomere (T2T) consortium
national human genome research institute
31
why sequence the human genome?
analyze genes for genetic mutations/diseases understand effects of genetic variation study human evolution develop personalized medicine
32
Steps to DNA sequencing
1. Nucleic acid hybridization base pairing to complementary strand 2. Determine nucleotide order specific gene sequence
33
Sanger sequencing
Frederick Sanger NP 1980 "Dideoxy sequencing" genes of interest dideoxynucleoside triphosphates anneal, fluorescent markers for each base contained
34
Adenine fluorescent marker
green
35
Thymine fluorescent marker
red
36
Guanine fluorescent marker
black
37
Cytosine fluorescent marker
blue
38
Next generation of DNA sequencing
"high throughout" Identify and amplify sequence of interest sequencing by synthesis computer reads nucleotides complements added real time
39
Current lab sequencers (availability)
over time has become much more affordable and efficient
40
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
41
Restriction enzymes
anti bacteriophage (virus for bacteria) mechanism restricts viral growth IDs and removes foreign DNA enzymes target specific restriction site
42
Methylation
protects self-DNA in bacteria against restriction enzymes
43
Restriction enzymes process
1. R enzyme cut at restriction site 2. plasmid and sequence of interest are cut 3. insert sequence between fragments 4. base pairing at new "sticky ends" 5. ligase seals new DNA-
44
Clustered regularly interspersed short palindromic repeats (CRISPR)
adapted from naturally occurring gene editing sys in bacteria (pro) target specific sequences for removal or replacement
45
endonuclease enzyme in CRISPR
1. Cas 9 (bacterial anti-virus) cuts DNA/inactivates viruses 2. specififc nucleotide guide sequence PAM (protospacer adjacent motif) has cut made adjacent cut made inside DNA
46
CRISPR system
1. target specific sequence in organism nuclease causes break in DNA 2. Knock OUT or IN 3. Compare to wildtype
47
Knock OUT CRISPR
mutate the gene -> inactivates
48
Knock IN CRISPR
insert new sequence into gene
49
DNA fingerprinting
small amount of DNA needed, PCR amplifies identifies person based on DNA
50
old DNA fingerprinting method
entire genome -> restriction enzymes different restriction sites -> DNA fragments use gel electrophoresis to create bands
51
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
52
FBI cultivates large ____ database
STR short tandem repeat profiling
53
Pharmacogenomics
drug effectiveness linked to gene sequence
54
Metagenomics
study of collective genomes multiple species interactions
55
Proteomics
entire set of proteins by cell study of proteome
56
Metabolomics
all small metabolites in organism based on genetic makeup study metabolomes
57
Which way does DNA flow in gel electrophoresis
Cathode to anode negative to positive
58
Why is Taq DNA polymerase used in PCR
because it doesn't denature at 72 C
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