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Biology 206- Genetics > Chapter 20 - Unit 4 > Flashcards

Chapter 20 - Unit 4 Flashcards

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1
Q

what is recombinant DNA technology

A

the use of in vitro techniques to isolate and manipulate DNA

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2
Q

what is an example of recombinant DNA technology

A

gene cloning

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3
Q

what is gene cloning

A

the process of isolating and making many copies of a gene in vitro or in vivo

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4
Q

does gene cloning only work on parts of DNA that are genes

A

no

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5
Q

what are restriction enzymes and what do they do

A

they are endocucleases that cut DNA and recognize restriction sites and are usually isolated from bacteria

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6
Q

what do DNA polymerases do

A

synthesize DNA; make DNA polymers

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7
Q

what do dNTPs do

A

they are AGCTs; needed for DNA polymerase to make a strand of DNA

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8
Q

what do ligases do

A

joins DNA together by phosphodiester bonds

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9
Q

what are cloning vectors and what do they do

A
  • they are any DNA molecule that is ammendible for inserting a GOI
  • the place where you insert the gene of interest
    includes; plasmids, and emulation of different DNA sources to make various things
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10
Q

how does gene cloning work with all of the “tools”

A
  1. isolate DNA (chrom.) from an organism or collection of cells
  2. Cut DNA and CV with same RE; insert (ligate) DNA fragments into CV to make recombinant DNA molecule
  3. Transform host cell w/ CV; host cell and recombinant CV replicate multiple times to generate identical copies of the GOI
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11
Q

every time that bacteria divides does the CV also divide

A

yes, there are times where the CV divisions outpace the host cell

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12
Q

what are the 3 possible outcomes from gene cloning in vivo

A
  1. host cell doesn’t transformed at all ( majority of bacteria)
  2. cell only gets CV (it was cut with RE but it ligated back upon itself)
  3. e.coli (or any) cell that has CV and its GOI is inserted (WHAT WE WANT)
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13
Q

what are the three ways REs can cut

A
  1. SmaI symmetrical
  2. BamHI asymmetrical
  3. PstI asymmetrical
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14
Q

how do SmaI cut

A

right down the middle and get 2 blunt ends

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15
Q

how do BamHI cut

A

staggered and get 5’ overhanging 5’ sticky ends; the two 5’ ends that are together can re-hybridize and come back together

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16
Q

how do PstI cut

A

staggered and get 3’ overhanging 3’ sticky ends; the two 3’ ends that are together can re-hybridize and come back together

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17
Q

what are REs that carry out asymmetrical cuts typically used in gene cloning

A

because it is more likely to get that hybridization b/w DNA and the CV?

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18
Q

what are E. coli plasmid

A

they are small circular DNA molecules

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19
Q

what are the four things that an E. coli plasmid CV must have

A
  1. origin of replication (ORI)
  2. multiple cloning sites (MCS)
  3. antibiotic resistance gene
  4. lacZ gene
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20
Q

what does the origin of replication do in E. coli plasmid CVs

A

it the region that allows the plasmids or CV to replicate inside of the bacterium, typically engineered to have enhanced replication rates; faster than normal

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21
Q

what does the multiple cloning site do in E. coli plasmid CVs

A
  • aka polylinker;
  • a region that has multiple restriction sites; depends on which RE you want to use; the other one you’re going to use w/ the other DNA molecule, its where GOI gets inserted
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22
Q

what does the antibiotic resistance gene do in E. coli plasmid CVs

A
  • ex: ampicilin
  • to distinguish b/w E. coli that got transformed w/ a CV and those that did not
  • bacteria on ampicillin w/ no CV and wasn’t transformed= die off
  • bacteria on ampicillin w/ CV & GOI and was transformed= survive
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23
Q

what does the lacZ gene (lacZ prime) do in E. coli plasmid CVs

A
  • codes for alpha fragments of beta gal.
  • alpha from CV and beta from bacterium= functional beta gal. to get blue pdt
  • no alpha from CV due to GOI in CV= no functional beta gal. can’t get blue pdt
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24
Q

what are the steps of the blue-white screening (gene cloning in vivo)

A
  1. cut CV with RE
  2. cut chromosomal DNA with same RE
  3. add ligase to create recombinant CV
  4. transform bacteria with recombinant CV and plate on agar containing ampicillin and X-gal
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25
Q

what are the three possible outcomes for blue-white screening

A
  1. recombinant CV; GOI inserted (WANT THIS)
  2. recombinant CV; no GOI inserted
  3. re-ligated CV
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26
Q

how do you stop a CV from being re-ligated (self-litigation)

A

add alkaline phosphotase (to take off P)

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27
Q

what is the purpose of ampicillin

A

to kill off cells that were not transformed

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28
Q

what is x-gal

A

it is a synthetic compound that beta gal. can convert into a blue pdt

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29
Q

what are the two ways of artificial transformation with blue-white screening.

A
  1. cold CaCl2 and heat shock

2. electroporation

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30
Q

what happens during cold CaCl2 and heat shock

A
  • get cool bacterial pellet and CaCl2
  • cold calcium will poke holes in the bacteria’s PM
  • then when you heat shock, it will create a draft bringing in those plasma molecules into the bacteria
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31
Q

what happens during electroporation

A
  • suspend bacterial pellet in steril h20, centrifuge and repreat
  • put competent cells into a a little tube
  • you apply electricity to poke hole in the PM to bring in the plasmids
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32
Q

what is colony hybridization

A

it is used to determine which colonies contain you GOI vs. some other chromosomal fragment

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33
Q

does colony hybridization use plates or gel

A

plates

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34
Q

what are the 4 step of colony hybridization (gene cloning in vivo)

A
  1. place a nylon membrane on the master plate and lift
  2. treat nylon membrane w/ detergent to lyse cells and the fix DNAinto
    membrane using UV light; add NaOH to increase the pH to denature DNA;
    add radio labeled probe (complementary to GOI)
  3. wash off unbound probe and expose membrane to x-ray film
  4. after you develop the film (autoradiogram), compare w/ master plate
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35
Q

what is the synthesis of complementart DNA (cDNA) used for

A

cDNA is used for cloning genes starting with mRNA instead of chromosomal DNA

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36
Q

what does cDNA stand for

A

complementary DNA

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37
Q

you should use the synthesis of cDNA when you want…….

A

to clone the coding sequence of the gene, not the entire gene

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38
Q

what are the conditions of the mRNA that are used to being the synthesis of cDNA

A
  • euk mRNA
  • has 5’ cap
  • has 0 introns
  • all coding sequences are the same
  • has poly A tail
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39
Q

what are the steps for the synthesis of cDNA

A
  1. add oligo (dT) primer= hybridize Ts to As from the poly A tail
  2. add reverse transcriptase to synthesize a stand of cDNA= extends the primer adding on the appropriate DNA nt
  3. get hybridized RNA & DNA; RNA will get partially digested with RNase H

4, add DNA polymerase I to synthesize second strand of DNA= replaces RNA nt with DNA nt

  1. add DNA ligase to seal gaps= get double-stranded cDNA
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40
Q

what is needed to do the polymerase chain reaction (gene cloning in vitro)

A
  • target DNA
  • two primers
  • taq polymerase
  • dNTPs
  • thermal cycler
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41
Q

what is the procedure of polymerase chain reaction (PCR) (gene cloning in vitro)

A
  1. denature at 95°C ( all H bonds are broken= ssDNA)
  2. anneal primers at 45-68°C (flank DNA of interest, not inside of it)
  3. extend primers at 72°C (by taq polymerase, and adds on appropriate dNTPs)
  4. repeat 20-40 times
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42
Q

when a round of PCR occurs do you get the double of what you stated with

A

yes

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43
Q

what is the advantage of PCR compared to in vivo

A

can get GOI in about 3-4 hours instead of 3-4 days

44
Q

to check that you got the correct PCR results what do you do

A

run it on a gel to see if it is placed correctly, if not that means there was some contamination

45
Q

why is reverse transcriptase-PCR (RT-PCR) used

A

it is used to detect the presence of a specific mRNA

46
Q

what is the two steps of RT=PCR

A
  1. synthesize cDNA from mRNA using reverse transcriptase (RT)
  2. amplify cDNA using PCR
47
Q

what are PCR and RT-PCR examples of and why

A

end point and semi quantitative procedures
- because they can do some quantitative measurements of intensity if bands in comparison it doesnt tell you how much you have; bc PCR isnt 100% efficient

48
Q

why is real-time PCR used

A

it is used to detect the quantitiy of DNA present or RNA expressed

49
Q

what is another name for real-time PCR

A

quantitative PCR (qPCR)

50
Q

what are the two steps in qPCR

A
  1. make cDNA deom mRNA in RT-PCR

2. carry out PCR with SYBR green or taqman-probe

51
Q

what happens if you carry out the PCR with SYBR green in qPCR

A
  1. denaturation
  2. primers annealed
  3. extension of primers- SYBR green gets in b/w the two DNA strands
  • more extension of DNA means more SYBR green florescence in b/w them
52
Q

what happens if you carry out the PCR with taqman-probe in qPCR

A
  1. denaturation
  2. primers annealed with taqman which is the polymerase; probe then hybridizes a portion of the DNA
    3, extension; taqman continues to move down the strand and it started to kick off the fluorescent pdt which will then flourece bc it wont be with the quencher which doesn’t allow it to glow
53
Q

what does southern blot do

A

it detects a particular gene sequence within a mixture of chromosomal fragments

54
Q

what does northern blot do

A

it detects RNA instead of DNA, if a particular gene is expressed, its mRNA (and any splice variants) can be detected

55
Q

Southern blot procedure (5 steps)

A
  1. cut DNA molecule with RE
  2. run DNA fragment on gel
  3. transfer DNA from gel to nylon membrane (blot)
  4. add a radio labeled probe which will hybridize to complementary sequence
  5. wash off any excess probe and expose membrane to x-ray film and audiogram
56
Q

northern blot procedure (4 steps)

A
  1. run RNA fragment on gel
  2. transfer DNA from gel to nylon membrane (blot)
  3. add a radio labeled probe which will hybridize to complementary sequence
  4. wash off any excess probe and expose membrane to x-ray film and audiogram
57
Q

RT-PCR southern blot

A

is just carrying out an RT-PCR then doing a southern blot

58
Q

RT-PCR vs. northern blot

A

RT-PCR is faster than norther blot and is more specific (want to see which sequence is being expressed)

59
Q

what is the procedure for RT-PCR southern blot

A
  1. do RT-PCR
  2. run that on gel (subjective to electrophoresis)
  3. follow up w/ a southern blot
60
Q

why is in situ hybridization (ISH) used

A

it is used to visualize gene activity directly in fixed cells or tissues

EX: fire and mello experiment with mex-3

61
Q

what could antisense oligonucleotides (ASO) do

A

they can be potential therapy for certain conditions (not reliable; long time ago; old)

62
Q

what is western blot used for

A

it is used to detect a specific protein from a mixture of proteins, and an antibody is sued as a probe

63
Q

western blot procedure

A
  1. get proteins from tissue/cell and dissolve them in SDS ( itll denature proteins by unfolding them or detergent coats protein wuth a negative charge); boil proteins to make sure they are all denatured
  2. separate them through electrophoresis (SDS-PAGE= polyacrylamide gel electrophoresis)
  3. transfer proteins onto nylon membrane (blotting)
  4. add 1° antibody that will bind to specific protein (POI) to wash unwanted things
  5. add 2° antibody (that is enzyme linked) to bind to 1° antibody
  6. add enzyme substrate from 2° antibody to produce colored/luminescent pdt
  7. expose it to x-ray film and develop autoradiogram
64
Q

what is dideoxynucleotide DNA sequencing procedure

A
  1. set up rxn “cocktail”
    • DNA template to be sequenced
    • radioactively-labeled primer to anneal to template
    • DNA polymerase (extend primer)
    • dNTPs
  2. divide mixture into 4 tubes, each “spiked” with a dideoxyNTP (ddNTP)
  3. start rxn
  4. when ddNTP randomly inserted into the growing strand, polymerization stops
  5. the rxn mixtures are run on a polyacrylamide gel and exposed to film (finner way to separate things)
  6. DNA bands are read bottom to top
65
Q

one dideoxynucelotide DNA sequencing has occurred once it continues to occur automatingly, what is that difference

A

the rxn occurs as before just this time in the same tube

66
Q

what is genetic engineering

A

it is the use of recombinant DNA technology to alter an organisms genotype and phenotype, and its application is called biotechnology

67
Q

what are inducible expression vectors

A

they are CV or plasmids that are designed to produce large numbers of a GOI mRNA to be translated and in that microbe
trying to express POI to get a lot of that protein

68
Q

why are inducible expression vectors used

A

we want to get the GOI expression to be regulated so that POI expression does not interfere w/ the growth of the host cell

69
Q

inducible expression vectors have a (strong/weak) promoter w/ a lac operon to regulate gene expression of GOI

A

strong

70
Q

what does the inducer do during inducible expression vectors

A

it binds to the repressor to knock it off the operator to continue what was started

71
Q

what is the inducer called that is used in inducible expression vectors and what does it do

A

isopropyl thygalatoside (IPTG) and it binds to the repressor to knock it off of the operator, so GOI can begin

72
Q

what is gene targeting

A

it is the process of altering a genetic sequence and observing the consequence in vivo

73
Q

why are knock-in (transgenic) organisms used

A

used to study the function of a gene when it is OVER-EXPRESSED in vivo

74
Q

what are genetically modified organisms (GMOs)

A

those that have received DNA from another species (transgene) and therefore are known as transgenic organisms

75
Q

why are knockout organisms used

A

used to study the function of a gene when it is UNDER-EXPRESSED in vivo (gene is not expressed at all)

76
Q

what does agrobacterium tumefaciens cause

A

it causes crown gall tumors in plants

77
Q

steps on how plants get crown gall tumors

A
  1. transfer DNA (T-DNA) is transferred from A. tumefaciens to infected plant cells and integrates into host plate chromosomes
  2. T-DNA produced growth hormones, causing uncontrollable cell growth, leading to crown gall disease
78
Q

what did we do to stop plants from getting crown gall disease

A
  • cut out the bad genes
  • replace it with the GOI that we want the plant to express
  • so that GOI will be transferred into plant cell
79
Q

what happens to the soy beans that are transgeneic and come in contact with round up

A

they are resistant to round up so they continue to grow

80
Q

what happens to the soy beans that are non-transgeneic and come in contact with round up

A

they die

81
Q

what do Bt toxins do

A

they paralyze the digestive tract of insect pests; harmless to humans

82
Q

what does golden rice 2 contain that white rice does not

A

a vitamin A precursor

83
Q

procedure of how to make trangenic mice

A
  1. inject several 100s-1000s copies of transgene into pronucleus of fertilized egg–> transgene will randomly insert into chromosome through nonhomologous recombination
  2. implant embryo into the foster mother
  3. embryo will express the transgene in all of its cells
  4. continue to breed to make transgeneic mice
84
Q

what are GloFish an example of

A

a transgeneic animal that was developed to detect toxins in the water

85
Q

what are “mad” transgenic rats an example of

A
  • transgene

- used to study aggressivness (aggressive protein is injected into mice)

86
Q

what is molecular pharming

A

it referes to the production of medically important proteins in:

  • agricultural plants (transgene plants)
  • mammary glands of lovestock (sheep, goats
87
Q

what does crispr do

A

REs cut at specific DNA sequence of phage DNA

88
Q

what is crispr cas 9

A

it is a genomic locus that is a bacterial form of adaptive immunity and it contains clustered regularly interspaced short pallindormic (read the same forward and backward 5’ to 3’ and vise versa) repeats

89
Q

what is the crispr mechanism

A
  1. spacer aquisition
    • phage gets cut into fragments which get inserted into the crispr loci
  2. crRNA biogenesis
    • crispr (ori transcribed and shortened to form crispr-derived RNA (crRNA)
  3. target interference
    • crRNA recruit a cas protein (nuclease) to cut invading phage of DNA
90
Q

crispr interference with type 2 steps

A
  1. cas9 selects spacer sequence flanked by photospacer adjacent motis (PAM)–> after cut, vital DNA fragment is inserted into crispr locus
  2. transactiviting scRNA (c tracer RNA)–> binds to complementart DNA
  3. cas9 domain-1 cut to for as double stranded breaks
91
Q

crispr interference with single guide RNA disrupt/KO steps

A
  1. combine cas 9 and sgRNA (crRNA + trace RNA)
  2. perform NHEJ repair (non homologous end joining)–> random patches
  3. end with indels that lead to framshift
92
Q

crispr interference with single guide RNA precise edit steps

A
  1. combine cas 9, sgRNA (crRNA + trace RNA), and transgene
  2. perform HDR repair (homologous directed repair)–> patches up homologous regions (less errors than other way)
  3. end with the insertion of the specific transgene
93
Q

what is xenotransplantation

A

when you put part of a species into another species (pig heart in human)

94
Q

what is the problem with PERV

A
  1. immunological rejection
  2. physiological incompatability
  3. microbes (ex: ERV–> immunodeficient sensor)
95
Q

what is PERV

A

same thing as ERV, just when in pigs its called Porcine endogenous retrovirus

96
Q

define population

A

group of individuals of same species that occupy the same region and can interpret

97
Q

define gene pool

A

all of the alleles of all individuals

98
Q

define population genetics

A

changes in genetic variation in a population over time

99
Q

how is allele frequency measured

A

number of copies of allele of interest (AOI) / the total number of alleles

100
Q

how is genotype frequency measured

A

number of individuals of genotype / total number of genotypes

101
Q

what does the Hardy-Weinberg principle state

A

states that the frequency of alleles in an ideal population will remain the same from genereationt o genreation (describes relationship b/w genotype and allele frequencies in a population)

102
Q

what are the conditions for an ideal population to be considered to be in hardy-weinberg equilibrium

A
  1. no. mutations in the GOI (not getting new alleles)
  2. no genetic drift (large population so allele frequency wont change that much
  3. no migration (no gene flow)
  4. no natural selection (all alleles and genotypes are successful in production and survival
  5. random mating
103
Q

if no population satifies all five conditions, do genotype and allele frequencies be approximated

A

yes

104
Q

in mammals, mare are __________ for X-linked genes, where are females have ____ copies

A

hemizygous, two

105
Q

among male, the frequency of any X-linked trait will ______ the frequency of males with the trait

A

equal

106
Q

how are female genotypes frequencies calculated

A

calculated using the hardy-weinberg equation

Biology 206- Genetics (4 decks)

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