Exam 3 (Final) Flashcards
(155 cards)
1
Q
In PCR, how many strands are copied?
A
Both strands
2
Q
Describe the general process of PCR.
A
1) Heat – denature template DNA and primers
2) Cool – anneal primers
3) Heat – DNA polymerase synthesizes new DNA
3
Q
Primers anneal best at ______(hot/cool) temperature.
A
cool
4
Q
DNA polymerases work best at ____(hot/cool) temperatures.
A
hot
5
Q
What end of the DNA primer has a component that allows nucleotides to be added to it? And what is the component?
A
3’-OH
6
Q
What does it mean if a vector or cell is “competent”?
A
It is able to take up foreign DNA
7
Q
Name three important components of vectors.
A
Origin of replication
Enzyme cut sites
Marker sequences
8
Q
When screening cultures (with the method that uses penicillin and B-gal) what color colonies are you looking for? What makes them that color?
A
Looking for WHITE colonies; made white when the insert is inserted into B-gal gene (turning off its expression of blue)
9
Q
When screening colonies, what special components are within the VECTOR and the INSERT that help in the screening process?
A
Vector – contains penicillin resistance and lacZ gene (makes cultures blue)
Insert – disrupts the lacZ gene, making that colony white
10
Q
If a colony grows blue, you know that it contains WHAT?
If a colony grows white, you know that it contains WHAT?
If a colony does not grow at all, you know that it contains WHAT?
A
If blue: contains vector but NOT insert
If white: contains vector and insert
If does not grow: contains neither
11
Q
If you want to determine fragment size, you should use WHAT?
A
Gel electrophoresis
12
Q
What does RFLP stand for? What is it? What can it show you?
A
Restriction Fragment Length Polymorphism; when two individuals show different patterns of band sized after enzyme digestion; Can show if there is a mutation in a organism, because the restriction enzymes will cut it differently
13
Q
Southern Blotting allows you to see WHAT?
A
If DNA contains a desired sequence; can also be used to compare DNA sequences across species/organisms
14
Q
In Southern Blotting, do you FULLY or PARTIALLY digest the DNA?
A
Fully
15
Q
In Southern Blotting, what kind of probe is typically used?
A
radioactive probe that expresses as a dark band when put against an x-ray film
16
Q
Differentiate between a genomic library and a cDNA library.
A
genomic library: contains full DNA
cDNA library: contains just isolated mRNA sequences (to see actively translating proteins)
17
Q
What roll does reverse transcriptions play in the creation of THIS SPECIFIC TYPE OF genomic library?
A
In a cDNA library, reverse transcription is used to turn mRNA into cDNA (complimentary DNA)
18
Q
When making a genomic or cDNA library, do you want FULL or PARTIAL digestion by enzymes? Why?
A
partial because you must use it to reconstruct the genome, using overlapping segments as your guide
19
Q
_______ can be used to scan genomic/cDNA libraries.
A
Southern Blotting
20
Q
What does FISH stand for? What is it?
A
Fluorescence in situ hybridization; used to see expression in specific locations (in site) in the body/tissue/organ/etc
21
Q
Dideoxy sequencing is also called WHAT?
A
Sanger Sequencing
22
Q
What is Dideoxy/Sanger Sequencing?
A
Using fluorescently labeled dideoxyribose (as well as normal deoxyribose), DNA elongation is cut short at different locations as it is replicated (in a similar manner to PCR, but only with ONE strand, because you only use a primer for one strand); end of nucleotide is labeled with a color; Perform gel electrophoresis and read out the sequence in order (from shortest segment to longest)
23
Q
What is it about dideoxyribose that makes it useful in sequencing DNA?
A
Missing an O on the 3’-OH (making it a 3’-H); This makes it unable to bind to the next nucleotide, halting elongation
24
Q
What is special about NEXT GENERATION SEQUENCING?
A
Relies on simultaneous sequencing of millions of partially digested fragments and then a massive reordering by looking at overlapping sequences
25
Genomic and cDNA libraries hold all the fragmented DNA in WHAT?
bacterial vectors
26
How do you read a microarray based assay that compares DNA from two different organisms?
cDNA from each organism is labeled with a different fluorescent color;
Your microarray "chip" has a bunch of different wells that contain complimentary binding sequences for a variety of known sequences; cDNA from the two organisms compete to bind with these sequences; if a well contains sequences that bind to cDNA in both organisms, the well will light up with a color that is in between the two colors you used to label your cDNA in each organism
27
What does PCR stand for?
Polymerase Chain Reaction
28
What does the ampR gene encode for?
enzyme beta-lactamase, which breaks down penicillin, enabling the bacterium to resist it
29
What are two types of NEXT GENERATION SEQUENCING?
Pyrosequencing and Illuminasequencing
30
What happens in PYROSEQUENCING?
- DNA to be sequenced in partially digested and made single stranded
- Each fragment is attached to a bead and placed in an individual well (similar to microarray)
- Each fragment is individually amplified within its well
- Tray is sequentially flushed with a particular type of deoxynucleotide triphosphate (A, T, C, or G), so that strands are synthesized in a controlled manner
- During each incorporation, 2 phosphates are cleaved off and provide the energy to generate a small flash of light
- All of this information can be integrated to sequence the DNA
31
What happens in ILLUMINA SEQUENCING?
- each base is assigned a uniquely colored fluor attached to a "stopper molecule" that prevents elongation until it is removed
- After each incorporation (similar to how it is done sequentially in pyrosequencing), a chemical reaction removes the fluor and "stopper molecule" and reads the residual flash of light, for which the color indicates the incorporated nucleotide
- Process repeated until all fragments are sequenced
32
Define germline mutation.
Present in either (or both) the sperm and the egg that made the individual, therefore present in every cell the individual has
33
Define somatic mutation.
Arise after fertilization, during division/replication/etc, thus only present in a subset of the individual's cells
34
Somatic mutations are passes down to how many of its descendents?
1/4th
35
What is a CONDITIONAL MUTATION?
Only arise under certain conditions
36
A food allergy is an example of a _________ mutation.
Conditional
37
How can somatic mutations be passed down to offspring?
If a mutation exists in the sex cells, it can be packaged into a gamete. This is a germline mutation from the perspective of the offspring.
38
How can strand slippage result in addition or deletion of nucleotides?
Newly synthesized strand loops out --> Addition
| Template strand loops out --> Deletion
39
Explain how repeated sequences can cause inversions and deletions.
Unequal crossovers results in a deletion on one strand and an addition on the other.
40
Define TRANSITION vs TRANSVERSION
Transition: purine-purine or pyrimidine-pyrimidine substitution
Transversion: purine-pyrimidine substitution (or vice versa)
41
Define MISSENSE vs NONSENSE mutation.
Missense: causes one amino acid to replace another
Nonsense: creates a stop codon at site of mutation
42
Define NEUTRAL mutation.
Changes amino acid content of protein but has no function consequence
43
Define SYNONYMOUS/SILENT mutation.
Does not change amino acid content
44
Define BENIGN POLYMORPHISM.
No change in protein activity.
45
How can synonymous mutations alter splicing?
- Can mutate within in the intron or exon
| - Almost all human introns begin with GT; so if C --> T mutation changes GC --> GT, the spliceosome may splice before GT
46
Most human introns begin with ____.
GT
47
When to trinucleotide repeats most often expand?
during cell replication/division
48
Define NON-replicative transposition vs replicative transposition.
Non-replicative: "cut and paste"
| Replicative: "copy and paste"
49
Describe the structure of a transposable element.
Terminal Inverted repeats flank the transposon.
50
What repeat sequence in generated after the transposable element inserts itself?
The flanking direct repeats
51
Do the flanking direct repeats come from the target DNA or the transposable element?
target DNA; they are generated from the staggered cuts that are made when the transposable element inserts itself
52
What is the role of transposase?
makes short, single-stranded overhangs on target DNA that are complimentary to each other; replicated to become the flanking direct repeats
53
What is a retrotransposon?
Transpose through an RNA intermediate using reverse transcription (more common in eukaryotes)
54
Describe the methods of a retrotransposon.
1) Retrotransposon DNA transcribed into RNA (DNA transposon is never removed from its sequence)
2) RNA undergoes reverse transcription to produce double-stranded DNA
3) Insertion proceeds as normal
55
Is retrotransposition a form of replicative or non-replicative transposition?
replicative, as the original DNA element is never removed from its location
56
Pigment production in grapes in controlled by a _________ element.
RETROtransposable
57
In relation to pigment production in grapes, when do the following colors come about?
BLACK
WHITE
RED
Black: fully functional gene
White: retrotransposon inserted into pigment gene
Red: retrotransposon moves out of the pigment gene after pigment production has already been halts for some time
58
What are the "Ac" and "Ds" elements?
Ac: ALLOWS the Ds element to move
Ds: what DISRUPTS (is inserted into) the pigment gene
59
What is the phenotype of each of these genotypes in corn?
Cc
Ctc
Ctc-->Ctc/Cc
Cc: Purple kernels
Ctc: Clear kernel
Ctc-->Ctc/Cc: Variegated kernel
60
How can transposable elements activate genes?
Transposable elements can have elements capable of acting as a promotor for a gene that it not within itself
61
In relation to causing chromosomal arrangements, pairing by LOOPING and crossing over between two transposable elements oriented in the SAME DIRECTION leads to ______.
Deletion
62
In relation to causing chromosomal arrangements, pairing by BENDING and crossing over between two transposable elements oriented in the OPPOSITE DIRECTIONS leads to ______.
Inversion
63
In relation to causing chromosomal arrangements, MISALIGNMENT and UNEQUAL CROSSOVER between transposable elements located on sister chromatids causes....
one chromosome with a deletion and one with a duplication
64
Define these three outcome of SUPPRESSOR MUTATION:
Double mutant
Intragenic
Intergenic
Double Mutant: a suppressor mutation at another site compensates from the original mutation and restores phenotype to wild type
Types of Double Mutants:
Intragenic: suppressor mutation within same gene's coding sequence
Intergenic: suppressor mutation within another gene's coding sequence
65
What is the different between a suppressor mutation and a reverse mutation?
Reverse Mutation: (reversion) replaces original mutation and restores WT genotype
Suppressor Mutation: at another site, but restores WT phenotype
66
What is normal: Protonated or NON-protonated bases? What is the difference?
Non-protonated is NORMAL. Protonated forms are ABNORMAL; have a shifted proton; this leads to mismatch and contributes to wobble
67
What type of reaction is depurination?
Hydrolysis
68
What types of bonds are cleaved in depurination reactions?
glycosidic bonds
69
What two nucleotides can be removed by deamination?
A and G
70
Describe how depurinations ultimately lead to mutations.
Depurination leaves a blank space where the purine nucleotide (A or G) should be; becomes a mutation when this depurinated strand is then used as a template strand; a purine (usually adenine) is incorporated into the new strand (instead of a pyrimidine).
71
What purine is usually inserted into the newly synthesized strand after a depurination of the parental strand?
Adenine
72
What occurs during deamination?
Removal of PYRIMIDINE's AMINO GROUP (NH2)
73
Ultimately, what change happens in the DNA that is synthesized down the line of strands that have been deaminated?
C-G base pairs change to T-A base pairs (a transition mutation)
74
Deamination of Cytosine makes _____, which binds with _____. Then, this nucleotide binds with _____.
Cytosine --deamination--> Uracil
U-A
A-T
75
Deamination of 5mC makes ______, which binds to _____.
5mC --deamination--> Thymine
| T-A
76
The most common type of transition mutation is....
C-G to T-A
77
What are BASE ANALOGS?
Look like one of the normal nitrogenous bases, but they BIND DIFFERENTLY.
78
What is an example of a base analog? What is it an analog for? What does is pair with?
5-Bromouracil is an analog for Thymine. Pairs with Guanine.
79
What is one process that makes reactive oxygen species?
Regular metabolism
80
What does an INTERCALATING AGENT do?
Insert itself between bases; distorts the shape of DNA double helix; often cause frame-shift mutations
81
What is a common consequence of intercalating agents?
frameshift mutations resulting in deletions
82
What does IONIZING RADIATION do?
ejects electrons and creates reactive ions and free radicals; often causes double stranded breaks
83
What is a common consequence of IONIZING RADIATION?
Double-stranded breaks
84
Attempts to fix mutations caused by THIS KIND OF MUTAGEN only cause more mutations.
Ionizing radiation
85
UV radiation causes ______.
Thymine (Pyrimidine) dimers
86
What is a pyrimidine dimer?
adjacent pyrimidines pull close together and cannot from hydrogen bonds with complementary bases
87
What is the ultimate effect of pyrimidine dimers?
blocks replication
88
________ have a special DNA polymerase that fixed pyrimidine dimers.
Eukaryotes
89
How can enzymes differentiate the newly synthesized DNA strand from the template strand?
New strand is briefly unmethylated
90
How is direct repair different from nucleotide base excision?
Direct repair FIXES the altered nucleotides; Nucleotide base excision REPLACES the altered nucleotides
91
What type of repair mechanism is photolyase involved in?
Direct repair
92
Describe how photolyase works.
Photolyase uses photoreactivation (in the dark; uses blue light) to rebind to pyrimidine dimers and cleave the joining bonds, re-allowing them to form hydrogen bonds with complimentary bases
93
How is base excision different from nucleotide excision?
In base excision, the base is removed first and THEN the sugar is removed. In nucleotide excision, the entire nucleotide is removed at once.
94
Describe what is happening in the ERROR PRONE process of base excision.
Eukaryotes use DNA polymerase beta to fill gaps, but it often messes up. Thus, AP endonucleases have to come back around (a second time) to remove the nucleotide and give DNA polymerase beta to fix it again. DNA polymerase beta does not have proofreading ability.
95
What happens in DNA damage bypass?
Newly synthesized (strand "S1") strand DOES NOT SYNTHESIZE over the damaged area on the template strand ("T1"). Synthesis (of "S2") continues on the other template strand ("T2"). Once area that corresponds to the damaged region (on "T1") is synthesized in "S2." The region of the template strand ("T2") that corresponds to the damaged area is relocated to the unsynthesized region of "T1." The new gap on "T2" is filled with DNA polymerase and DNA ligase.
96
What are the two (kind of like three) ways in which double-stranded breaks can be repaired?
```
Nonhomologous recombination
Homologous recombination (using either the newly synthesized strand OR the homologous strand as a template)
```
97
What differentiates the two types of homologous recombination in double-strand repair?
In both types, the 3' of the DNA with the gap invades the non-gapped homolog. This is used to synthesize ONE HALF (one strand) of the double-stranded DNA molecule with the gap. The other strand with the gap can be filled in TWO WAYS:
- Use newly synthesized strand as template
- Use other side of homolog (the one that was not already used) as template
98
what percent of human genome has come from transposable elements?
~45%
99
What is an example of TIME DEPENDENT gene regulation?
Globin genes
100
What does the basal transcription apparatus bind to? What does it do?
core promotor; allows for basal (minimal) rates of transcription
101
How are eukaryotic repressors different from those in prokaryotes?
Eukaryotic repressors do NOT directly block RNA polymerase. Instead they:
- compete with activators
- bind to near sites and interact from a distance
- interfere with assembly of basal transcription mechanism
102
Enhancers are limited by ______.
Insulators
103
What does an insulator bind to?
The enhancer
104
Where must an insulator be in order to "insulate"?
Between enhancer and promotor
105
Enhancer binds to the ______.
Promotor
106
What does DNAse 1 do?
Cut DNA non-specifically; opens up everything so that binding sites can be accessed
107
What makes an area more sensitive to DNAse 1?
highly active transcriptionally
108
Lysines have what charge?
Positive (+)
109
Acetylation does what to DNA?
opens it up by adding negative acetyl groups to positive lysines
110
Lysines are found on _____.
Histones
111
Methylated binding proteins have two domains. What are they?
1) methylated DNA binding regions
2) domain with histone deacetylase activity
- --> Tighten DNA
112
Methylated DNA binding proteins serve what purpose?
Tighten up DNA
113
How does MECP2 assert its function?
it is a methylated DNA binding protein; represses transcription
114
Do twins have different patterns of methylation?
Yes
115
What does royal jelly do to female bees?
silences DNA methyltransferase and ALLOWS TRANSCRIPTION of genes that will make her develop into a queen
116
What is a PARAMUTATION?
Heritable epigenetic change
117
Describe how alternative splicing in flies can determine gender.
alternative splicing of Tra gene mRNA by sex-lethal (sxl) gene regulates sex determination; Sxl is ACTIVATED in FEMALES; Tra and Tra2 work together to splice Dsx in females and make them female
118
From what end to RNAses begin to degrade mRNA? How is this problem fixed?
5'; poly-A-tail helps to stabilize mRNA so that it can remain in the cytoplasm to be translated
119
What are the two types of iRNAs? What make them the same?
miRNA and siRNA;
BOTH come from longer, DOUBLE-STRANDED RNA precursor that is cleaved in CYTOPLASM by Dicer.
Join with proteins to make RISC (RNA-induced silencing complex).
have some complementary sequence to the mRNA that they will degrade
120
What are the two types of iRNA? What makes them DIFFERENT?
miRNA: endogenous (transcribed in cell), SINGLE-STRANDED
siRNA: exogenous (taken up by cell), DOUBLE-STRANDED
121
What are the three mechanisms by which iRNA asserts its effects?
- inhibition of TRANSLATION (binds IMPERFECTLY to mRNA and prevents ribosome from attaching)
- inhibition of TRANSCRIPTION (attach to DNA + attract methylating enzymes)
- cleavage of mRNA (iRNA pairs completely with target mRNA)
122
How does lincRNA-p21 affect transcription?
lincRNA-p21 represses action of transcription factor p53, which regulates the cell cycle and apoptosis, by attracting iRNAs
123
What happens in WILSON'S DISEASE? What can be used to treat it?
copper is not cleared from the body because Metallothionine (MT) gene is hypoactive; ZINC can be used in INDUCE the MT gene
124
What also rises if concentration of heme is increased in a cell?
globin production also rises --> combine to form hemoglobin
125
Inducer can knock off ________.
Repressor
126
What can knock of the repressor?
The inducer
127
_______ bind to silencers.
Repressors
128
What are the two types of homologs?
Orthologs: Found in two different species
Paralogs: Found in a single organism
129
Interspersed repeated elements are often due to.....
transposable elements
130
What is METAGENOMICS?
Identifying the sequences of all the organisms in an environment (such as in a soil sample)
131
HYBRIDIZATION STUDIES can be used to study WHAT?
How similar two species' DNA is
132
Describe what happens in a hybridization study.
1) DNA from two species is denatured and fragmented
2) Allowed to reanneal
3) Similarity in sequences assessed by the temperature that is required to re-denature the newly annealed double strands
133
What are two types of DNA that do not undergo recombination?
mitochondrial DNA (mDNA) and the Y chromosome
134
What benefits to FADS have and what can be said of its allelic diversity though evolution?
FADS allow you to make poly saturated fats (that the brain needs) from the food that you eat; high frequency of high-activity FADS found in early humanoids that migrated away from water
135
FOXP2 gene is instrumental in what function?
Speech
136
HAR1 serves what function in development?
Development of cerebral cortex
137
Differences in the number of CNVs of the amylase gene reflect...
Different amounts of starch in that population's typical diet
138
What is COLINEARITY or SYNTENY?
order of genes conserved between species
139
What is an example of "third generation" sequencing?
Nanopore Sequencing
140
Positional genetics is also called __________, because...
forward genetics; because you FIRST locate an abnormal phenotype from which you want the sequence from and THEN you look for mutations.
141
In positional cloning, once you isolate your gene of interest, you can perform _________ to detect changes in expression in those affected vs unaffected.
RT-PCR (reverse transcriptase PCR)
142
What makes "reverse genetics" reverse?
You induce mutations and THEN look for abnormal phenotype.
143
Transgentic animals are an example of ________ (forward or reverse?) genetics.
Reverse; because you induce the mutation and then look for affects
144
Describe the process by which TRANSGENIC ANIMALS are created.
1) One of the two pronuclei are injected with foreign DNA, and you hope this induces the desired mutation(s)
2) Embryos are inserted into a pseudopregnant surrogate mother and allowed to develop
3) Breed the progeny that have been bred with the defective gene (variegated animals)
4) Hope that you create homozygous knockouts
145
Genetic engineers have made plants that produce _______ proteins, so protect them against predators. What is the effect of these proteins?
Cry proteins; cleaved by gut enzymes into small proteases that bind to gut epithelial cells and disrupt the osmotic balance (enhancing inward water flow)
146
Explain how an inserted vector can protect mRNA from degradation.
Vector used to insert a "protection mechanism" into another organism. For example, the rspT element (which comes from the 3' UTR of the tobacco plasmid 16s, which is a promotor for one of the most actively transcribing genes) can be added to other mRNA sequences so that it can help enhance the translation of an mRNA sequence. RpsT forms a looped structure at the 3' end of mRNA to protect it from degradation.
147
Describe the components necessary for "pharming" human AAT in sheep's milk, and say what organism they originate from.
- Coding Sequence: from HUMAN AAT gene (so human system will later accept the AAT)
- Promoting Sequences: from SHEEP udder cells (because the available transcription factors will all be from the sheep)
- Localizing Signal: Allow for modifications that make it possible for AAT to be secreted into the sheep's milk
148
Transfection vs Transduction
Transfection: use methods (shock, heat, etc) to make cell more receptive to uptake foreign DNA; commonly use liposomes as vessel
Transduction: uses a VIRUS to transfer DNA that has been augmented with a desired DNA sequence; viruses infect cells by injecting DNA/RNA, which both ultimately work through DNA
149
Uses a VIRUS: Transfection or Transduction?
Transduction
150
Makes cells MORE RECEPTIVE to foreign DNA: Transfection or Transduction?
Transfection
151
How did a few instances of gene therapy gone wrong create leukemia?
Construct integrated into a stem cell in such a way that it activated a gene that promotes cell replication and division
152
Use _____ to flank the construct that you wish to integrate.
Homologous Recombination
153
Describe the roles of neo+ and tk+ in relation to how they can be used in methods of gene knockout.
1) Neo+ inserted in middle of target gene (the gene you want to knock out) to disrupt its function
2) Disrupted target gene is flanked by homologous recombination sites that correspond to area of the "target chromosome" into which you want to insert the KO gene
3) Construct made with tk+ just outside one of the homologous recombination sites
4) In culture, the disrupted target gene (with tk+ and Neo+) is allowed to interact with DNA from the organism you wish to insert it into
5) Successful transfers contain neo+ but NO tk! (neo+, tk-_)
154
When scanning for successful KO gene incorporation, what do you look for?
Successful transfer is (neo+, tk-)
- Neo+ confers neomycin resistance
- tk- cells with be able to grow in gancyclovir (tk+ cells will die in gancyclovir)
Grow cells in medium containing neomycin and gancyclovir, and only the successful transfers will survive.
155
Cell Mediated vs Direct Delivery
Cell Mediated: uses STEM CELLS
| Direct Delivery: uses a VIRUS or RETROVIRUS to inject foreign DNA
