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foundations of biology > ch 20.1-20.4: biotechnology and genomics > Flashcards

ch 20.1-20.4: biotechnology and genomics Flashcards

1
Q

recombinant DNA technology

A

allows researchers to recombine specific DNA sequences from any organisms

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2
Q

what does recombinant DNA create?

A

DNA molecules that are not found in nature

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3
Q

DNA cloning

A

producing many identical copies of a gene or other DNA sequence

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4
Q

where is the cloned sequence of DNA inserted?

A

into a plasmid

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5
Q

plasmid

A

small circular pieces of DNA found in bacterial cells

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6
Q

restriction endonucleases

A

cuts DNA molecules at specific base sequences for insertion into a cloning vector

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7
Q

where are DNA molecules cut?

A

specific DNA sites called recognition sites

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8
Q

what seals the DNA back together after being cut?

A

DNA ligase

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9
Q

cloning vector

A

a modified plasmid or virus used to transfer recombinant genes into cells

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10
Q

genetically modified organisms

A

plant or animal that has specific changes introduced to its DNA using genetic engineering methods

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11
Q

what are GMOs used for?

A
  1. research purposes
  2. generating crops and domestic animals with desired traits
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12
Q

gene knockout

A

a loss of function mutation that most often has been engineered rather than naturally occurring

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13
Q

what are 2 ways to knockout genes?

A
  1. deduce function of the gene by observing what happens when the gene does not work
  2. modify the gene
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14
Q

what can GMO animals with altered genes be used to determine?

A

where and when in development a gene product is expressed

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15
Q

to produce GM plants, what is created?

A

transgenic plants

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16
Q

process of making a transgenic plant

A
  1. tumor-inducing genes from T-DNA region of Ti plasmid are taken out and replaced with genes they want to introduce to the plant
  2. engineered Ti plasmid is used to transfer the recombinant plasmid DNA into the plant and now the introduced genes are a part of the plant cell’s genome
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17
Q

T-DNA genes

A

genes in the plant genome where they promote uncontrolled plant cell growth

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18
Q

gene therapy

A

treatment of an inherited disease either by introducing a normal form of the gene or correcting an improperly functioning gene

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19
Q

what is needed for gene therapy to work?

A

disease that is due to defects in a single gene and sequence of wild-type allele must be known

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20
Q

how does gene therapy work?

A

therapeutic allele is introduced into affected individuals and express in the correct tissues, in the correct amount, and at the correct time

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21
Q

how are therapeutic genes delivered?

A

genes are packaged into genetically engineered viruses (vectors) for transport into human cells

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22
Q

what if there is a dominant disease allele when performing gene therapy?

A

there must be a way to replace defective allele with one that functions normally

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23
Q

how are therapeutic genes delivered during gene therapy?

A

by vectors

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24
Q

vectors

A

genetically engineered viruses
- genomes have been altered to allow the incorporation of the therapeutic genes and disable replication of the virus in target cells

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25
what can vectors not do?
they cannot replicate the virus in the target cell but they can still gain access to the cell to deliver the gene
26
two approaches of gene therapy
1. ex vivo 2. in vivo
27
ex vivo
cells that require therapeutic gene to be removed from patient and then infected with the viral gene therapy vector which integrates the therapeutic gene into the patient's genome - outside the body
28
example of ex vivo gene therapy
CAR-T cells are engineered outside the patient's body and infused into the patient where they recognize and work to destroy cancer cells
29
in vivo
vector is injected into bloodstream and the virus is transported throughout the patient's body - inside the body
30
somatic cell gene therapy
genetic modification of non-reproductive cells
31
heritable gene therapy
genetic modification can be transmitted to reproductive cells and future generations
32
CRISPR-Cas
genome editing method based on snRNA associated with a Cas protein that cuts DNA at sites complementary to the sgRNA
33
CRISPR locus
compromised of repeat sequences separated by spacer sequences derived from viruses that have infected the cell or its ancestors - DNA sequences that protect against viral infections
34
process of CRISPR locus
1. CRISPR locus is transcribed into a long pre-crRNA 2. pre-crRNA is processed into short fragments called crRNA 3. crRNA binds to complementary DNA of an invading virus along with a short noncoding RNA called tracrRNA 5. crRNA and tracrRNA are bound together by Cas protein
35
Cas protein
enzymes that cut DNA complementary to a crRNA
36
what does each crRNA contain?
spacer region and an RNA copy of a viral genome with short stretches of bacterially encoded repeat sequences
37
sgRNA
chemically synthesized DNA that could be transcribed in vitro to create a fusion of crRNA and tracrRNA
38
process of sgRNA
1. sgRNA is mixed plasmid that contained the gene for Cas9 and introduced into the nucleic acids of a cell 2. sgRNA guides Cas9 to a complementary target sequence in the genome 3. Cas9 makes cuts in the DNA
39
what does the Cas9 endonucleease do?
make double-stranded cuts in the DNA
40
nonhomologous end joining
double-stranded DNA break can be repaired by an error-prone mechanism that joins the broken DNA back together, usually with insertion or deletion
41
what happens if the DNA is cut in a coding sequence of a gene?
the reading frame will be disrupted after repair
42
what happens if DNA is cut in a regulatory sequence of DNA?
the regulatory sequence will be altered
43
homology-directed repair
intact DNA is exchanged for broken DNA - the intact DNA is introduced in the cell using CRISPR-Cas machinery
44
which CRISPR-Cas genome editing method is used to disrupt gene function?
nonhomologous end joining
45
which CRISPR-Cas gene editing method is used for DNA modification?
homology-directed repair
46
polymerase chain reaction (PCR)
technique that rapidly generates many identical copies of a specific stretch of DNA
47
what is needed for PCR?
primers
48
primers
short lengths of single-stranded DNA that match sequences on either side of the region to be amplified
49
what are the 3 steps of PCR
1. denaturation 2. annealing 3. extension
50
denaturation
separates two strands of DNA
51
annealing
attaches primers to DNA
52
extension
synthesizes complementary DNA strand from dNTPs, starting at primer
53
why are primers needed for PCR?
they allow DNA synthesis reaction to begin
54
after each round of DNA amplification in PCR occurs, what happens?
number of target DNA doubles
55
DNA fingerprinting
identifying individuals based on their unique genome using short tandem repeat sequences
56
what do eukaryotic genomes have?
short tandem repeats: short DNA sequences repeated along chromosome - number of STRs differ in each individual
57
process of DNA fingerprinting
1. obtain DNA sample 2. PCR 3. gel electrophoresis
58
what does the gel electrophoresis determine?
number of repeats - fewer repeats, the shorter the PCR product
59
quantitative reverse transcriptase PCR
form of PCR where RNA is converted to cDNA using reverse transcriptase, then PCR is used to amplify the cDNA in cycles that is monitored for the amount of amplified cDNA
60
cDNA
DNA sequence created from mRNA using reverse transcriptase
61
environmental DNA (eDNA)
helps ecologists learn which species exist in an environment - collected and amplified
62
how to infer evolutionary relationships?
comparing sequences of the same gene in different species
63
dideoxy sequencing
uses chemically modified nucleosides (ddNTPs) in combination with normal nucleosides (dNTPs)
64
what happens when ddNTPs are used?
they stop synthesis of DNA when added by DNA polymerase to a polynucleotide chain because ddNTP has no hydroxyl group at the 3' position
65
how is the sequence of the ddNTPs determined?
by detecting the terminal 3' base at each position in the DNA
66
what are the components of the Sanger DNA sequencing reaction?
1. DNA polymerase 2. short primer complementary to the template strand 3. all 2' deoxynucleoside triphosphates 4. all 2' and 3' dideoxynucleoside triphosphates that are labeled uniquely so each can be detected 5. template DNA
67
what is important in the Sanger DNA sequencing reaction?
the ratio of ddNTPs and dNTPs because ddNTPs make the strand shorter
68
high-throughput sequencing
used to simultaneously sequence many different template molecules
69
how does high-throughput work?
by physical binding of template DNA to a solid surface or to microbeads and amplification of DNA templates by PCR
70
shotgun sequencing
involves sequencing many copies of genome in short fragments
71
what happens in shotgun sequencing?
many copies of genome are randomly broken up and the fragments can overlap - overlap regions are used to assemble the genome
72
do sequencing methods produce short or long reads?
short reads - Sanger: 750 bases -Next Gen: 150 bases
73
functional genomics
identify and annotate function to various parts of the genome
74
open reading frames
DNA sequence that contains codons for amino acids with no stop codons and may encode parts of proteins
75
comparative genomics
compare genomes both within and across species
76
gene annotation
identifying genes or other functionally important sequences
77
how many reading frames are possible on each strand?
3 reading frames, so a total of 6 in one DNA molecule
78
what do bioinformatic programs look for to find genes?
1. start codons 2. sequences for promoters 3. ribosome binding sites 4. other regulatory sites
79
what happens after the open reading frame is found?
sequence is compared to known gene sequences from other species - similarities are due to homology
80
why is looking for open reading frames in eukaryotes more complicated?
reading frames are broken up by introns - not possible to scan for long ORFs
81
gene-finding strategy for eukaryotic genomes
1. isolate mRNA and make cDNA from it using reverse transcription 2. sequence the cDNA (expressed sequence tag) 3. search for matches between the EST and a sequence of DNA
82
why is reverse transcription needed to find genes in eukaryotic genomes?
to remove introns
83
genome wide association study
numerous SNPs and bioinformatics allow rapid searches for markers associated with disease
84
genetic markers
genes or other loci have known location
85
what is needed to work as a genetic marker?
1. DNA sequence must come from at least 2 common sequence variants 3. single nucleotide polymorphisms: site in DNA that varies in a single base pair
86
where are SNPs usually located?
outside of coding or regulatory regions and they have no direct effect on phenotype
87
how is an SNP used to identify a trait?
if the trait and SNP allele almost always occur together, the gene for the trait is near the known location of the SNP
88
mendelian traits
single gene, affecting discrete phenotypic differences

foundations of biology (25 decks)

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