Exam 4 Flashcards
(185 cards)
1
Q
Growth hormone
A
- produced by pituitary systems and regulates body size
- gigantism or dwarfism can be attributed to GH excess or deficiency
2
Q
whether genetic variation is deemed pathological or benign part of human diversity…
A
depends upon perspective
3
Q
Production of hGH in the 1950s
A
- GH deficiency was treated by injected by cadaver-derived human GH
- pool pituitary glands have GH
- GH extracted with Wilhemi preparation
- c-hGH is injected into hGH deficient humans
4
Q
drawbacks of c-hGH
A
- requires a lot of cadavers bc method only produces TINY amounts of hGH
— prevented clinical testing for additional treatment uses
— many people were denied due to scarcity
5
Q
organismal clones
A
exact genetic copies of entire organism
6
Q
cellular clones
A
groups of genetically-identical cells
7
Q
molecular/DNA clones
A
identical molecules (e.g. DNA)
8
Q
hgH protein biotechnology
A
1) isolate the DNA of the hGH gene
2) clone the gene – make many copies of the hGH gene in vivo
3) Make bacteria transcribe and translate the gene to make a ton of hGH
9
Q
Restriction digest VS PCR for isolating genes?
A
PCR
10
Q
Restriction digest
A
- chops up one genome into millions of restriction fragments
- still don’t know where the gene is and may only have a few copies total in the sample
11
Q
PCR
A
- allows us to make many copies of the exact sequence we want
- replicate target DNA into may copies (2^n)
(n = number of cycles)
12
Q
what does PCR gel look like?
A
- large dark band show many copies of the amplified DNA region
- no band for the template (genomic) DNA on gel because there is not enough present to be seen
13
Q
Defining primers of a specific region
A
- Primer is the exact same sequence as the 5’ to 3’ next to the region of interest (on both sides)
- The PCR product will extend from the 5’ end of one primer to the 5’ end of the other
14
Q
Although PCR allowed for isolation of the gene of interest….we are still not ready to put GH gene into bacteria
A
- When bacteria replicates, linear DNA will be lost
- need cloning vector that will be replicated when cell replicates DNA during mitosis
15
Q
3 regions of a plasmid
A
- origin of replication
– so plasmid can be replicated - multiple cloning site (MCS)
– many different restriction enzyme sites so things can be inserted into plasmid (often LacZ gene) - antibiotic resistace gene
– any cells that uptake this plasmid will be resistant to a particular antibiotic
16
Q
digestion with EcoRi
A
- circular plasmid is cut at EcoRI sequence leaving sticky ends and EcoRi sites on both sides
17
Q
problems with c-hgH
A
- c-hGH was contaminated with prions
- gave many people Creutzfeldt-Jakob disease
- CJD is the human version of mad cow
—- incurable neurodegenerative disease (years to develop and lethal)
18
Q
EcoRI restriction sites are added to the…
A
5’ end of primers for isolation of the region of interest
19
Q
Isolation of GH gene by PCR and engineering of EcoRi sites on either side…
A
- EcoRi addition creates identical sets of sticky ends on both the plasmid and GH gene
- sticky ends by EcoRi allows for recombination and GH gene to be added to the plasmid after annealing of primers and ligase
20
Q
Why is ligase needed for recombination?
A
seals the backbone after sticky ends are bound
21
Q
recombinant plasmid DNA
A
- transformed into bacteria which replicate it with its endogenous DNA replication machinery
- the plasmid replicates with the bacteria and we now have a lot of recombinant molecules inside the bacteria if transformation worked
- even if it did…there may be some untransformed bacteria mixed in!!!
22
Q
antibiotic selection
A
is used to identify bacteria that contain the plasmid
23
Q
antibiotic selection plates with VS without colonies
A
colonies = transformants (have plasmid)
no colonies = non-transformants (no plasmid)
24
Q
problem just looking at the colonies
A
- know the Amp-resistant colonies have a plasmid but we do NOT know if they have a plasmid that contains the GH gene
25
what may cause the plasmid to form without GH gene in it?
- sticky ends come back together without GH getting in!
- plasmid would have Amp resistance gene but not GH gene
26
what is the significance of having a MCS located in a lacZ coding gene?
B-galactosidase activity can be visualized using X- gal (blue colored reporter gene)
*If B-gal is functional, X-gal is converted into a blue chemical
- MCS doesn't disrupt LacZ alleles and functional B-gal is produced (blue color)
- If gene is inserted at MCS, it does disrupt LacZ allele so NO B-gal is produced (white color)
27
LacZ functional
blue color
(no gene insertion at MCS)
28
LacZ nonfunctional
white color
(gene insertion at MCS)
WE WANT WHITE (select for white)
29
The strain of E.coli must be LacZ-...
so functional lacZ is from the plasmid
30
Steps of creating recombinant DNA
1) genomic DNA
PCR
2) hGH gene and restriction sites + cloning vector (plasmid)
digest
3) complementary sticky ends pair
ligate
4) final vector
5) transform into bacteria
6) look for white colonies
31
Gel with antibiotic selection and white-blue screening
1) pick colonies
2) grow each colony in individual culture
3) Isolate plasmid DNA
4) Cut with EcoRi
5) Run gel
Blue: one band of DNA
White: one DNA plasmid band and one band for GH gene
--- GH gene travels farther bc it is smaller than plasmid DNA
32
Gel with antibiotic resistance (not blue/white assay)
1) Pick colonies
2) Grow each colony in individual culture
3) Isolate plasmid DNA
4) Run PCR with primers specific to GH gene
one large band representing the PCR product
33
Steps for isolating the GH gene
1) isolating GH gene via PCR and engineer in EcoRI sites with primers
2) Ligated GH fragment into plasmid that is cut with EcoRi at MCS.
3) Verify that we have some transformant clones with GH gene through: antibiotic selection & blue-white screening + digest gel (PCR)
34
3 basic steps for GH gene
1) isolate DNA of hGH gene
2) clone the gene (make many copies in vivo)
3) make bacteria transcribe/translate the gene to make more hGH gene
35
regulatory element allows for the bacteria to express the hGH gene
promoter
- prokaryotic if end goal is to make protein in bacteria
(bacterial RNA polymerase won't recognize eukaryotic promoter)
- same for eukaryotic promoter
- prokaryotic and eukaryotic ==> recombinant DNA
36
hGH coding gene is inserted downstream of the Lac promoter .... why?
1) allows hGH to be expressed in bacteria under control of lac regulatory elements
2) enables blue-white screenign (only if inserted LacZ)
3) our cloning vector could function as a bacteria expression vector
37
Why is mature mRNA way longer than it is supposed to be in transformed bacteria?
- prokaryotes don't splice (so introns are still present)
- they do not have the proper machinery to splice a eukaryotic gene
38
Why is cDNA library used instead of genomic DNA?
- cDNA does not contain introns and will have a shorter length of bp
- no extra material
39
Using cDNA for gene expression in bacteria
1) PCR: amplify GH cDNA from pituitary cDNA library with primers in restriction sites
2) Ligate GH fragment into vector that is cut with same RE and has prokaryotic promoter
3) Select colonies grown on Amp (use BW test also)
4) colonies that have GH gene should be tested for protein expression
5) GH protein can be grown in large vats and GH is purified from media
40
GMO
- Genetically Modified Organisms
- recombinant organism
- any organism that contains DNA that has been recombined from multiple sources
41
Why do some people not support GMOs? ""
- can pass transgenic DNA to you
- unanticipated ecological effects if released into the environment
- Organic foods are more nutritious
- There is not a consensus among scientists (though 88% believe they are safe)
42
Papaya GMOs
- modified to express a single protein from papaya ringspot virus (confers resistance to contracting the virus
43
transgene is no different from...
any other gene
44
AquaAdvantage salmon
- growth hormone gene promoter is typically only active at certain times of the year
- GH gene promoter is replaced with a continually active promoter
- ecological impact is carefully monitored
- strict requirements about releasing salmon/eggs into the environment
45
GMO golden rice
- GMO rice is modified to express the B-carotene (vitamin A)
- could be considered more nutritious especially in areas where vitamin A deficiency is prevalent
46
GMO soy
- soy was engineered to express a protein from Brazil nuts to cause an allergic reaction in people who are allergic to Brazil nuts
- NOT approved by food safety authorities
47
GMO corn
- corn borers are small caterpillars that eat and destroy corn
- farmers often spray corn with Bacillus thuringiensis toxin (Bt)
- toxin considered an organic pesticide when purified directly from bacteria that normally produce it
- Bt toxin unfortunately degrades in the sun and other concerns about affects on pests/bugs
- INSTEAD...
-- Agrobacterium tumefaciens can insert pieces of its Ti plasmid into plant genomes
-- Recombinant DNA allows plants to have new trait eliminating the need for pesticide
*** typically pathogenic but Ti plasmid is altered to remove harmful part and still include gene of interest
48
steps for creating GMO corn
1. isolate DNA from the Bacillus thuringiensis
2. Amplify (PCR) the Bt toxin gene
3/4. Digest (RE) vector/plasmid and digest gene of interest
5. Create recomb. DNA molecule
6. Transform corn plant
7. Select for transformed Agrobacteria
8. Transform corn plant
9. Select for transformed corn plants
49
functions of a vector
1. a cloning vector to be replicated and selected for in bacteria (agrobacterium)
2. serve as an expression vector once in plants
3. carry info for "selection" of transformed plants
50
if we want to express gene in eukaryote...
eukaryotic promoter must be upstream the gene of interest
- RNA polymerase does not recognize a prokaryotic promoter
51
antibiotic resistance genes will have ____ promoter
prokaryotic
52
CRISPR-Cas9
bacterial defense system against viruses.
- Bacteria integrate bits of viral genetic material into their genomes.
- encode for RNAs that can bind to viral genomes by complementary base pairing, allowing the bacteria to detect and destroy viral DNA
**cut DNA at precise location using guide RNA and Cas9
53
Cas9
enzyme that cuts DNA to form single-stranded breaks
54
cutting DNA with CRISPR
- guide RNA directs Cas9 to cut at specific locations based on base pairing
55
How is the bacterial CRISPR-Cas9 system modified to be simpler to use for biotechnology purposes?
- created a single guide RNA which functions the same as 2 seperate RNAS (sgRNA)
56
CRISPR-Cas9
- specific and programmable enabling targeted genome sequencing
57
specific
Cas9 will only cut at a particular DNA seqeunce
58
programmable
The DNA sequence it cuts is not set in stone, but rather can be determined (by the sequence of the guide)
59
why wouldnt restriction enzymes be particularly useful for targets genome editing?
- restriction enzymes have specific DNA sequences and are NOT programmable
Ex/ EcoRi will always cut at a specific point (GAATTC)
*cannot be used for targeted gene sequencing
60
PAM site
A short DNA sequence, the protospacer-adjacent motif (PAM), is frequently used to mark proper target site
NGG
(N= any nucleotide)
* found in the non-complementary strand
*N is adjacent to 3' end of guide on the opposite strand
61
2 conditions must be met for Cas9 to recognize and cut DNA:
1) the single guide RNA base pairs to the DNA
2) The DNA in the non-complementary strand contains a PAM sequence in the correct location (directly adjacent to where the guide binds, on the side closest to the 3' end of the guide)
62
where is the PAM sequence found?
- strand no complementary to the sgRNA
- 3' end of sgRNA in other strand would be adjacent to complement of NGG (5'-CCN-3')
5'-NGG-3')
63
sgRNA and PAM
- sgRNA does not contain the PAM
- sgRNA found right before PAM
- sgRNA contains the same sequence (Us instead of Ts) as the bottom strand (where PAM is found)
64
if the guide contained the PAM...
that part of the guide would also base pair to the top strand
65
2 ways that cells normally repair double-stranded breaks:
1) non-homologous end joining (NHEJ)
2) homology directed repair (HDR)
66
NHEJ repair
- repair double-stranded break by doing 2 ends directly together
* could ADD or REMOVE nucleotides to the sequence first before ending joining
67
How does CRISPR-Cas9 edit DNA?
- causes a DSB that is incorrectly repaired at a precise location
- using NHEJ: a random change (insertion/deletion) will be introduced at a precise location
68
NHEJ and CRISPR-Cas9
- random change (indel) will be introduced at the targeted site
- if break is repaired correctly, it will be recognized by sgRNA and cut again!
(used to create knockouts/loss-of-function mutations)
*random mutation is likely a loss-of-function mutation
-Two cells edited with the same guide could end up with a different repair.
(null, hypomorphic, silent)
69
HDR repair
break is repaired using homologous chromosome as template to ensure proper sequence
- homologous template "fills in the blank" of what was lost
70
HDR and CRISPR
-relies on homologous chromosomes/sister chromatids
-- UNLESS scientists provide a repair template with homology arms (regions of homology to the L/R of break site); the cell can be used instead
**used to generate gene knock-ins
71
gene editing can be used to generate
transgenic organisms
72
transgenic model mouse
1) mouse embryonic stem (ES) cells are genetically modified
2) edited stem cells are injected into a blastocyst
(cells are pluripotent and totipotent
3) mosaic pups are born
(cells are mix of original blastocyst cells and edited stem cells)
4)mosaic pup are crossed to WT
73
blastocyst
early stage mouse embryo
hollow ball of undifferentiated cell
74
pluripotent and totipotent
- cells can develop into any kind if cell type given the proper cues
P: adult
T: embryo and adult (all stages)
75
mosaicism
occurs when a person has two or more genetically different sets of cells in his or her body.
76
transgenic mouse phenotypic ratios
- 0% homozygous for edited DNA
- 25% homo for transgene
- 50% hetero for transgene
- 25% homo for WT
* always heterozygous for edited gene bc only female or male is affected most often
77
how to get mice homozygous for edited genes?
- selfing of edited gene heterozygotes
50/50: heterozygotes/homozygous dominant
78
problems with transgene mice
- germline may be derived from the blastocyst rather than the ES cells injected
--- if no pups inherit the genome edit when crossing WT and mosaic
79
hemoglobin
- tetramer of 4 protein subunits (2 of each kind)
- protein in red blood cells that carries oxygen
- each subunit is encoded by its own gene
80
hemoglobin subunits
Adult: 2 alpha-globin and 2 beta-globin
Fetal: 2 alpha-globin and 2 gamma-globin
81
mutations in beta-glopbin gene
cause 2 serious blood disorders:
1) sickle cell disease and protein aggregation
2) beta-thalassemia
82
what causes SCA?
- specific missense mutation changes the 6th amino acid from glutamate to valine
83
what causes beta-thalassemia?
- any null/amorphic mutations that reduces the amount of b-globin produced
84
changes in globin expression from fetus to adult
- around birth, y-globin expression mostly turns OFF and b-globin expression is turned ON (along with continual production of alpha globin)
85
CRISPR-based gene therapy for treatment of SCA and b-thalassemia
- BCL11A is a TF that promotes the switch from expressing y-globin(fetal) to b-globin(adult)
-Casgevy introduces mutations in an ENHANCER that normally turns BCL11A on after birth
- gene that produces BCL11A has its own promoters and enhancers that are controlled by other TFs!
- Casgevy mutates an enhancers of the BCL11A gene so that it is not made and affects how the globin genes are regulated
- preventing BCL11A expression means that y-globin can be expressed
86
BCL11A
TF that promotes the switch from expressing y-globin(fetal) to b-globin(adult)
87
How does BCL11A affect the expression of various globin genes?
- in fetuses, a TF/coactivator/enhancer interaction promotes y-globin expression
- Around birth, another TF (BCL11A) starts being expressed.
-- when it binds to y-globin promoter, it prevents the activating complex from forming
----- the activating complex instead forms on the b-globin promoter
88
transcription factors
- regulate gene expression
- TFs themselves are encoded by the genes
- genes that produce TF have its own promoters and enhancers that are regulated by other TFs
89
mutations of the BCL11A gene enhancer
- prevents BCL11a expression
- allows y-globin to be activated and expressed
--- activating complex can once again form on the promoter of the y-globin gene and drive expression
*** increase of fetal hemoglobin after editing
90
how does hemoglobin editing work?
- patient's own bone marrow cells are edited
- process similar to a person given a bone marrow transplant to themselves
*** cells are edited between donating marrow and getting the marrow transplant
91
steps of hemoglobin editing via bone marrow transplant
1) stem cells from bone marrow are extracted
2) stem cells are genetically modified and BCL11A gene is turned OFF
3) Genetically engineered SCs are transplanted back to bone
4) SC make healthy fetal hemoglobin and normal RBCs
92
advantages to the hemoglobin SC engineering strategy
1) causing a gene knockOUT is easier than a gene knockIN --> it doesn't not require HDR with a template (you just break the gene)
2) Casgevy works for multiple kinds of b-globin disorders regardless of what specifically is wrong with a particular patient's B-globin gene
3) using a patient's own (edited) marrow cells makes rejection of a transplant unlikely
93
Sanger sequencing elements
- DNA synthesis for Sanger sequencing includes ddNTPs, so synthesis will stop whenever one is added even though only 1 primer is used
94
ddNTP
- dideoxynucleotide triphosphates
- N = any bp
- Has no 2' or 3' OH (can be added to chain but no additional synthesis would occur)
95
if only ddNTPs added were ddGTPs...
- the sequence would terminate at G when ddNTP is incorporated
- fragment lengths reflect base position in sequence
96
gel electrophoresis
separates fragments by size
- repeating with all 4 ddNTPs reveals the nucleotide at each position (line up all 4 reactions to read up the sequence ladder)
97
using separate lanes or fluorescent labeling
- can show which ddNTPs tag a fragment
- can show the order of base pairs in a single lane
98
the first base pair on the 5' end of a sequence (on a gel)
bottom of the gel and moves up
--- shortest bc added first
99
modifications to Sanger sequencing
- capillary gel electrophoresis (used laser through DNA sample and detector)
- automated detection (sequence analysis and reconstruction on computer)
100
current sequencing methods generate...
reads that are 100s or 1000s of base pairs long
101
read
the sequence determined by a single sequencing reaction
102
how big is a genome?
e.coli - 5 Mb
humans - 3.1Gb
P. japonica - 149 Gb
D.melanogaster - 180 Mb
103
genomes and any other DNA sequences too long to sequence in its entirety must be...
assembled from reads that are small enough to actually sequence
104
contig
a set of DNA segments/sequences that overlap in a way that provides contiguous (touching) representation of a genomic region
105
assembling contigs requires...
identification of overlapping reads
(reads are like sentence fragments that need to be assembled into contigs like sentences)
106
the human genomes was first sequenced in ...via...
2000s
- competition and collaboration between the government (Human Genome Project) and the private sector (Celera Genomics)
107
reference genomes
tools that give people a common reference to compare to
analogy -- fully assembled triceratops skeleton for reference to other dinosaurs
108
"the" human genome?
a fully assembled refernce
--- not an individual genome
(what commonalities are found in all human genomes)
109
a reference genome is NOT...
- the genome of a single individual
- synonymous with wild type or normal
- 100% perfectly assembled or complete (it may improved over time!)
110
annotating a genome involves....
- finding and labeling functional elements such as enhancers, promoters, genes, etc.
* instead of a giant list of billions of letters, you know what parts of the genomes correspond to specific functional elements
111
bioinformatics tools and databases allow genomes...
to be computationally explored and compared
112
New "next generation" sequencing methods have...
increased sequencing speed and decreased cost (by nearly 1 million)
113
can u identify a causative mutation by checking all the bases that differ from the reference genome?
NO there will be millions of differences to check because the genome is so BIG
- extracting useful into from genome data remains a challenge
- most of the human genome is shared yet we still have a tremendous amount of genetic variation
114
can u identify a causative mutation (a specific disease) in a genome sequence?
- MAYBE...we can check what differences we can find in the regions of the genome that have been annotated as the disease gene or one of its known regulatory elements
115
The human genome is extremely variable so...
it may be easy OR hard to tell which is causative
EX/ easy to tell -- deletion of enhancer promoter and coding region from a single SNP
versus causative difference that is not obvious
116
many SNPs occur in only ___ or ___ versions...
2 or 3 versions
(T,G,C,A)
117
SNPs
- common differences between genomes (single nucleotide variant will be considered a SNP if it becomes a frequency of at least 1%)
118
How is a SNP different from a substitution?
- every SNP begins as a substitution mutation (rare variant that becomes COMMON)
119
what types of SNPs could change the sequence of a protein?
located in the protein coding region of a gene (EXON)
- not introns or intergenic regions
120
SNPs may or may not be the...
cause of a mutant phenotype
121
SNPs that do not cause a mutant phenotype...
may still be useful as linked markers
122
linked SNPs
no effect on protein production or function (just bc a SNP is in an exon does NOT mean it must be causative)
123
Causative SNPs
- in the regulatory region = changes the amount of protein produced
- in coding region/protein = changes amino acid sequence
124
When 2 DNA sequences are located close together on a chromosome...
- it is unlikely that a meiotic crossover will occur in between them --- so they are unlikely to independently assort (LINKAGE)
125
ideal "marker SNP"
one that is completely linked to whatever causes the trait of interest (recombination frequency is ~0%)
126
Associations between easily genotyped SNPs
- take advantage of known associations between easily genotyped SNPs and whatever linked variant is responsible for a phenotype even if we do NOT know what the causative nearby variant !!!
*may not know what allele is responsible for the trait, but linked SNPs can sometimes still help us make predictions about phenotype
(haplotype presence can show commonality)
127
Genotyping linked "marker" SNPs act as...
- the basis for commercial genetic testing
- some causative SNPs are tested but linked SNPs are included too
---- bc we often do not know what causes certain traits!!
** In many cases, it may involve the effects of multiple genes, which complicates things further
128
haplotype
- sets of SNPs that do not independently assort (inherited as one unit
* many SNPS are completely linked to all other nearby SNPS
129
recombination hot spots
- SNPs not located in recomb. hot spots are almost never separated from each other by recombinations
- not all regions of the genome are equally likely to undergo recombination
130
calculating the possible number of haplotypes
base pair possibilities ^ SNPS
ex/ 2 bp possiblities with 3 SNPs = 8 haplotypes
* but only some are observed in the population
131
haplotype observations in populations
- only some haplotypes are observed in a population
- new haplotypes could arise in a population if recombination occurred in the right place
- BUT the SNPs are so close together (and not in recomb. hot spot) that the probability of this happening is 0%.
132
How to use SNPs to determine certain genetic outcomes
1. Locate SNPs in a region of DNA
- (examine DNA in and around gene)
2. Create haplotype group (create groups with certain SNPs and determine people that fall into certain groups)
3. Test haplo response to certain condition/treatment
133
genetic testing
- help people make informed decisions about their health/reproduction
- help patients understand how to interpret results from genetic testing
134
PGD
- pre-implantation genetic diagnosis tests embryos for genetics traits before implantation in uterus
1. mothers eggs are collected
2. each is fertilized with sperm
3. fertilized eggs are placed on Petri dish to grow
4. embryos divide for 3 days
5. blastomere is removed from each embryo
6. blastomere is tested to see if its embryo contains the defective gene carried by one or both parents
7. defective gene discarded or donated to reserach
8. good genes are implanted or frozen for later use
135
using genetics to control reproduction is a -- and -- not a --- question
- ethical and philosophical question NOT a scientific question
- bc history shoes us that genetics can be used a tool or a weapon (nazi sterilization)
136
Eugenics
immoral and pseudoscientific theory that claims it is possible o perfect people and groups through genetics and the scientific laws of inheritance
- use incorrect and prejudiced understanding of Charles Darwin and Gregor Mendel to support the idea of "racial improvement"
137
when should fingerprint of human DNA be used?
- determining if a suspect was at the scene of a crime using willingly provided by the suspect (not forced or secretly)
- attempting to exonerate people wrongfully convicted of crimes
- identifying remains from people killed in a natural disaster or war
- paternity testing
138
DNA finger printing
- relies on highly variable SSR loci.
- 13 different loci are tested
- each person has 2 alleles for each locus and there are many differently sized alleles for each locus the population
*** fingerprinting looks at the sizes of PCR products made from all 13 loci
139
DNA profile gel electrophoresis
- size standard added to every lane
- other lanes are fluorescently labeled PCR products
- compare the fragments to see if relatives DNA fingerprints match relatives
* babies have STR loci from each parent
140
restriction enzymes
bacterial proteins that recognize specific, short nucleotide sequences and cleave the DNA backbone at those sites; generates restriction fragments
141
digestion
process of cleaving/cutting DNA
142
blunt ends
an end of the dsDNA molecule that has no 5' or 3' overhang
143
sticky ends
breaks phosphodiester bonds on 2 strands of the DNA molecule at offset locations
-- results in a single protruding strand at each end (about 4 bases long) that are free to base pair with other DNA cut by the SAME restriction enzyme
144
gel electrophoresis
separates DNA fragments according to size
-- uses marker fragments to determine migration distances of traveling bands (size of band --- related to distance)
145
molecular cloning
the process by which a single DNA fragment is purified from a complex mixture of DNA molecules and then amplified into a large number of identical copies
146
cloning vectors
vehicles for introducing foreign DNA into host cells, where the DNA can be reproduced in large quantities; DNA molecule into which another DNA fragment of appropriate size can be integrated without the loss of the vector's capacity for replication
147
DNA clone
a group of identical replicated DNA molecules
148
recombinant DNA molecules
a combination of DNA molecules with parts having different origins that were joined using recombinant DNA technologies
149
2 Characteristics for sticky ends for the efficient production of vector-insert recombinant
1. single-stranded overhands available for base pairing
2. 2 sticky ends produced with the same ensue,es are always compatible (complementary in sequence)
150
plasmids
small circles of dsDNA that can replicate in bacterial cells independently of the bacterial chromosome (commonly used as cloning vectors)
151
selectable markers
vector genes that make it possible to identify cells harboring a recombinant DNA molecules
152
colony
a mound of genetically identical cells that all descend from a single cell
153
genomic library
a collection of DNA clones clones that together carry a representative copy of every DNA sequence in the genome of a particular organism
154
PCR
replicates DNA sequences in vitro by amplifying DNA products of each previous round of replication
- PCR primers defines the ends of the target region
155
reverse transcriptase
a retroviral RNA-dependent DNA polymerase that synthesizes DNA strands complementary to an RNA template
* copies a single strand of RNA into a strand of cDNA (complementary DNA)
156
cDNA library
a large collection of cDNA clones that represent the mRNAs expressed by a particular cell type, tissue, organ, or organism
157
genomic VS cDNA library
genomic: represent all regions of DNA equally and show what the intact genome looks like in the region of each clone
cDNA: reveal which parts of the genome are transcribed in specific tissues and how those transcripts are processed into mRNAs
158
proteome
complete set of proteins encoded by a genome
159
T-DNA
the parts of the Ti plasmid of Agrobacterium tumefacians that integrates into the host plant genome
--- T-DNA used as a vector for generating transgenic plants
160
CRISPR
- cluster regularly interspaced short palindromic repeats
- region in many bacterial genomes that confers immunity to viral infection
- Cas proteins associated with CRISPR
161
CRISPR/Cas9 system
a genetically engineered version of the immunity system of streptococcus progenies that is used for genome editing
- sgRNA brings Cas9 to target site in the genome
-Cas9 makes double-strand breaks in the DNA
- results in knock outs or knock ins through DNA repair
1. ssRNA (single guide RNA)
2. Cas9 protein
162
targeted mutagenesis
technology that enables scientist to alter any particular base pairs in the genome
(gene editing, knockins/outs, gene editing with CRISPR)
163
gene targeting
method for inserting DNA into a genome that relies on homologous recombination; the DNA is targeted for insertion into a specific place in the gnome by sequence similarity
- mutagenize a specific gene intro and then introduce the mutant DNA Into bacterial cells
- homologous recomb then replaces the normal copy of the gene in the bacterial genome with the mutant copy
164
embryonic stem cells (ES)
cultured embryonic cells that continue to divide without differentiating and are capable of becoming any cells type
165
2 obstacles for gene-targeting in eukaryotic organism
1. gene targeting must occur in germ-line cells
2. must screen a large number of germline cells to obtain one with the desired mutation
166
totipotent
cells have the ability to produce every type of cell found in the developing embryo and adult animal
* cell state early in embryonic development
167
chimera/mosaic
an organism composed of cells originally from 2 or more different organisms
168
knockout
homozygous for an amorphic allele of a gene induced by gene targeting
169
knockin
gene has been altered by targeted mutagenesis
--- new DNA is introduced with an altered DNA sequence that is knocked in (exon)
170
Sanger sequencing depends on...
DNA polymerase
171
DNA polymerase requires
1.) ssDNA template
2.) deoxyribonucleotide triphosphate (building blocks for newly synthesized DNA)
3.) primer (short ssDNA molecule complementary to part of template and provides free 3' end for bonding)
172
sanger sequencing method
1. hybridization of template and primer
2. generation of a nested set of fragments
3. dideoxynucleotide structure does not let DNAP add anymore nucleotides
4. products analyzed on gels
5. analyze image of sequencing gel with single/multi lanes and dyes
173
read
in a single DNA sequencing run, a digital file of sequence of As, Gs, Cs, Ts constituting the newly synthesized DNA
174
contigs
stretches of contiguous DNA sequence obtained by virtual alignment of sequence reads; each chromosome is a contig
(humans have 24 contigs)
175
bioinformatics
the science of computational methods -- specialized software to decipher the biological meaning of information contained within organisms
176
species reference sequence
a single complete annotated version of a species genome
- genome browsers show the arrangement and structure of genes with RSQ
177
BLAST
rapid, automated matching of particular DNA/amino acid sequences across multiple sequence for analytic of evolutionary relationships
178
DNA polymorphisms
2 or more alleles at a locus
- the sequence variations of a DNA polymorphism can occur at any position on a chromosome
179
anonymous polymorphism
differences in a genomic DNA sequence with no effect on gene function
180
nonanonymous polymorphism
differences in genomic DNA sequence that have an effect on gene function
181
DNA marker
identifiable physical location on a chromosome with DNA sequence variants whose inheritance can be monitored
182
SNP (single nucleotide polymorphism)
single nucleotide locus with 2 naturally existing alleles defined by a single bp substitution
-- SNP loci are useful as DNA-based markers for genetic linkage analysis
183
PGD
prenatal genetic diagnosis
- genotyping fetal cells to determine allies to disease loci
184
cell-free fetal DNA analysis
procedure where DNA in the blood of pregnant people is analyzed to show fetus genotype
---- DNA related from broken fetal cells that have leaked into the bloodstream
185
DNA fingerprint
a multi-locus pattern produced by the detection of SSR loci
**AKA DNA profile
