Chapter 20 - Unit 4 Flashcards
(106 cards)
1
Q
what is recombinant DNA technology
A
the use of in vitro techniques to isolate and manipulate DNA
2
Q
what is an example of recombinant DNA technology
A
gene cloning
3
Q
what is gene cloning
A
the process of isolating and making many copies of a gene in vitro or in vivo
4
Q
does gene cloning only work on parts of DNA that are genes
A
no
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
6
Q
what do DNA polymerases do
A
synthesize DNA; make DNA polymers
7
Q
what do dNTPs do
A
they are AGCTs; needed for DNA polymerase to make a strand of DNA
8
Q
what do ligases do
A
joins DNA together by phosphodiester bonds
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
10
Q
how does gene cloning work with all of the “tools”
A
- isolate DNA (chrom.) from an organism or collection of cells
- Cut DNA and CV with same RE; insert (ligate) DNA fragments into CV to make recombinant DNA molecule
- Transform host cell w/ CV; host cell and recombinant CV replicate multiple times to generate identical copies of the GOI
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
12
Q
what are the 3 possible outcomes from gene cloning in vivo
A
- host cell doesn’t transformed at all ( majority of bacteria)
- cell only gets CV (it was cut with RE but it ligated back upon itself)
- e.coli (or any) cell that has CV and its GOI is inserted (WHAT WE WANT)
13
Q
what are the three ways REs can cut
A
- SmaI symmetrical
- BamHI asymmetrical
- PstI asymmetrical
14
Q
how do SmaI cut
A
right down the middle and get 2 blunt ends
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
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
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?
18
Q
what are E. coli plasmid
A
they are small circular DNA molecules
19
Q
what are the four things that an E. coli plasmid CV must have
A
- origin of replication (ORI)
- multiple cloning sites (MCS)
- antibiotic resistance gene
- lacZ gene
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
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
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
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
24
Q
what are the steps of the blue-white screening (gene cloning in vivo)
A
- cut CV with RE
- cut chromosomal DNA with same RE
- add ligase to create recombinant CV
- transform bacteria with recombinant CV and plate on agar containing ampicillin and X-gal
25
what are the three possible outcomes for blue-white screening
1. recombinant CV; GOI inserted (WANT THIS)
2. recombinant CV; no GOI inserted
3. re-ligated CV
26
how do you stop a CV from being re-ligated (self-litigation)
add alkaline phosphotase (to take off P)
27
what is the purpose of ampicillin
to kill off cells that were not transformed
28
what is x-gal
it is a synthetic compound that beta gal. can convert into a blue pdt
29
what are the two ways of artificial transformation with blue-white screening.
1. cold CaCl2 and heat shock
| 2. electroporation
30
what happens during cold CaCl2 and heat shock
- 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
31
what happens during electroporation
- 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
32
what is colony hybridization
it is used to determine which colonies contain you GOI vs. some other chromosomal fragment
33
does colony hybridization use plates or gel
plates
34
what are the 4 step of colony hybridization (gene cloning in vivo)
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
35
what is the synthesis of complementart DNA (cDNA) used for
cDNA is used for cloning genes starting with mRNA instead of chromosomal DNA
36
what does cDNA stand for
complementary DNA
37
you should use the synthesis of cDNA when you want.......
to clone the coding sequence of the gene, not the entire gene
38
what are the conditions of the mRNA that are used to being the synthesis of cDNA
- euk mRNA
- has 5' cap
- has 0 introns
- all coding sequences are the same
- has poly A tail
39
what are the steps for the synthesis of cDNA
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
5. add DNA ligase to seal gaps= get double-stranded cDNA
40
what is needed to do the polymerase chain reaction (gene cloning in vitro)
- target DNA
- two primers
- taq polymerase
- dNTPs
- thermal cycler
41
what is the procedure of polymerase chain reaction (PCR) (gene cloning in vitro)
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
42
when a round of PCR occurs do you get the double of what you stated with
yes
43
what is the advantage of PCR compared to in vivo
can get GOI in about 3-4 hours instead of 3-4 days
44
to check that you got the correct PCR results what do you do
run it on a gel to see if it is placed correctly, if not that means there was some contamination
45
why is reverse transcriptase-PCR (RT-PCR) used
it is used to detect the presence of a specific mRNA
46
what is the two steps of RT=PCR
1. synthesize cDNA from mRNA using reverse transcriptase (RT)
2. amplify cDNA using PCR
47
what are PCR and RT-PCR examples of and why
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
why is real-time PCR used
it is used to detect the quantitiy of DNA present or RNA expressed
49
what is another name for real-time PCR
quantitative PCR (qPCR)
50
what are the two steps in qPCR
1. make cDNA deom mRNA in RT-PCR
| 2. carry out PCR with SYBR green or taqman-probe
51
what happens if you carry out the PCR with SYBR green in qPCR
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
what happens if you carry out the PCR with taqman-probe in qPCR
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
what does southern blot do
it detects a particular gene sequence within a mixture of chromosomal fragments
54
what does northern blot do
it detects RNA instead of DNA, if a particular gene is expressed, its mRNA (and any splice variants) can be detected
55
Southern blot procedure (5 steps)
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
northern blot procedure (4 steps)
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
RT-PCR southern blot
is just carrying out an RT-PCR then doing a southern blot
58
RT-PCR vs. northern blot
RT-PCR is faster than norther blot and is more specific (want to see which sequence is being expressed)
59
what is the procedure for RT-PCR southern blot
1. do RT-PCR
2. run that on gel (subjective to electrophoresis)
3. follow up w/ a southern blot
60
why is in situ hybridization (ISH) used
it is used to visualize gene activity directly in fixed cells or tissues
EX: fire and mello experiment with mex-3
61
what could antisense oligonucleotides (ASO) do
they can be potential therapy for certain conditions (not reliable; long time ago; old)
62
what is western blot used for
it is used to detect a specific protein from a mixture of proteins, and an antibody is sued as a probe
63
western blot procedure
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
what is dideoxynucleotide DNA sequencing procedure
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
one dideoxynucelotide DNA sequencing has occurred once it continues to occur automatingly, what is that difference
the rxn occurs as before just this time in the same tube
66
what is genetic engineering
it is the use of recombinant DNA technology to alter an organisms genotype and phenotype, and its application is called biotechnology
67
what are inducible expression vectors
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
why are inducible expression vectors used
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
inducible expression vectors have a (strong/weak) promoter w/ a lac operon to regulate gene expression of GOI
strong
70
what does the inducer do during inducible expression vectors
it binds to the repressor to knock it off the operator to continue what was started
71
what is the inducer called that is used in inducible expression vectors and what does it do
isopropyl thygalatoside (IPTG) and it binds to the repressor to knock it off of the operator, so GOI can begin
72
what is gene targeting
it is the process of altering a genetic sequence and observing the consequence in vivo
73
why are knock-in (transgenic) organisms used
used to study the function of a gene when it is OVER-EXPRESSED in vivo
74
what are genetically modified organisms (GMOs)
those that have received DNA from another species (transgene) and therefore are known as transgenic organisms
75
why are knockout organisms used
used to study the function of a gene when it is UNDER-EXPRESSED in vivo (gene is not expressed at all)
76
what does agrobacterium tumefaciens cause
it causes crown gall tumors in plants
77
steps on how plants get crown gall tumors
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
what did we do to stop plants from getting crown gall disease
- 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
what happens to the soy beans that are transgeneic and come in contact with round up
they are resistant to round up so they continue to grow
80
what happens to the soy beans that are non-transgeneic and come in contact with round up
they die
81
what do Bt toxins do
they paralyze the digestive tract of insect pests; harmless to humans
82
what does golden rice 2 contain that white rice does not
a vitamin A precursor
83
procedure of how to make trangenic mice
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
what are GloFish an example of
a transgeneic animal that was developed to detect toxins in the water
85
what are "mad" transgenic rats an example of
- transgene
| - used to study aggressivness (aggressive protein is injected into mice)
86
what is molecular pharming
it referes to the production of medically important proteins in:
- agricultural plants (transgene plants)
- mammary glands of lovestock (sheep, goats
87
what does crispr do
REs cut at specific DNA sequence of phage DNA
88
what is crispr cas 9
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
what is the crispr mechanism
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
crispr interference with type 2 steps
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
crispr interference with single guide RNA disrupt/KO steps
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
crispr interference with single guide RNA precise edit steps
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
what is xenotransplantation
when you put part of a species into another species (pig heart in human)
94
what is the problem with PERV
1. immunological rejection
2. physiological incompatability
3. microbes (ex: ERV--> immunodeficient sensor)
95
what is PERV
same thing as ERV, just when in pigs its called Porcine endogenous retrovirus
96
define population
group of individuals of same species that occupy the same region and can interpret
97
define gene pool
all of the alleles of all individuals
98
define population genetics
changes in genetic variation in a population over time
99
how is allele frequency measured
number of copies of allele of interest (AOI) / the total number of alleles
100
how is genotype frequency measured
number of individuals of genotype / total number of genotypes
101
what does the Hardy-Weinberg principle state
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
what are the conditions for an ideal population to be considered to be in hardy-weinberg equilibrium
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
if no population satifies all five conditions, do genotype and allele frequencies be approximated
yes
104
in mammals, mare are __________ for X-linked genes, where are females have ____ copies
hemizygous, two
105
among male, the frequency of any X-linked trait will ______ the frequency of males with the trait
equal
106
how are female genotypes frequencies calculated
calculated using the hardy-weinberg equation
