Midterm Flashcards
(100 cards)
1
Q
The Fly Lifecycle
A
~10 days in 25 degrees Celsius
- 1 day: embryo
- 1 day: 1st instar larva
- 1 day: 2nd instar larva
- 1 day: 3rd instar larva
- 2 days: prepupa
- 2 days: pupa (cocoon)
Fully grown male or female
2
Q
The Drosophila Genome
A
~ 160 Mb of DNA
18,000 genes
- Diploid organisms
3
Q
How many chromosomes do drosophila have? How many are autosomes and how many are sex chromosomes?
A
- Drosophila have 4 chromosome :
X or Y, 2, 3, and 4 (with 4 being very tiny) - 2, 3, and 4 are autosomal
- X or Y are sex chromosomes
4
Q
WT phenotypic markers
A
- Red eyes
- Long bristles
- Brown/tan body
5
Q
Stock 103 flies
A
Curled wings up
6
Q
Stock 107 flies
A
Smaller eye-shape and blunt, short bristles
7
Q
Genes can be given names that correspond with …
A
Mutant phenotypes (i.e., white for white eye color). The symbol for a mutant allele is an abbreviation of the gene name, such as w for white eyes
8
Q
Alternatively, genes are given CG numbers…
A
(conceptual gene) numbers (i.e., CG7456) if the gene was first inferred from the sequence of the genome rather than a phenotype
9
Q
Genes often have multiple…
A
names because they have been discovered independently by different means. Gene names or symbols of any type are always printed in italics
10
Q
Gene names and symbols are always printed in
A
Italics
11
Q
For genes named by mutant phenotype, a “+” signifies…
A
The wild type allele
- For example, white (w) is the allele for the white gene in Drosophila. w indicates the mutated allele and w+ indicates the WT allele
12
Q
When named after a phenotype, lowercase indicates…
A
That the mutant allele is recessive to the WT allele.
13
Q
Homologous chromosomes are separated by a…
A
Slash
14
Q
When named after a phenotype, upper case letters indicate a…
A
Mutant allele is dominant to a WT allele
ex. A Cy/Cy+ heterozygote has the CY (curly) wing phenotype
15
Q
Homologous chromosomes and the X/Y chromosomes are separated by slashes (/). If the slash is missing…
A
Assume that the two homologous copies of the chromosome are identical ( w v), assume that the two homologous copies are identical. For example, e/e+ indicates a heterozygous, while just e indicates homozygous e/e
16
Q
Genes on the same chromosome are separated by…
A
Spaces. For example, w v/w+ v+ indicates that w and v genes are on the same chromosome (the X chromosome)
17
Q
A semicolon (;) separates…
A
separates genes on non-homologous chromosomes. Chromosome order is X/Y; 2nd; 3rd; 4th. For example, w/w+ ; e/e+ indicates a fly heterozygous for white on the X chromosome and heterozygous for ebony on the 3rd chromosome. Chromosomes that are WT are not listed (2nd and 4th chromosomes are WT in this strain).
18
Q
The Y chromosome is not…
A
Italicized and is always written as a capital letter
19
Q
If gene symbols are written between brackets (for example, [y+ attP])…
A
It means that the corresponding genes or DNA sequences have been introduced as a transgene
20
Q
If nothing is written for an allele, gene, or chromosome…
A
Assume it is WT
21
Q
Try the example problems at the end of the first lecture
A
22
Q
Remember to add semicolons between chromosomes in “Full Chromosome notation”
A
23
Q
Remember that the Y chromosome is denoted as a hook
A
24
Q
Components of the CRISPR Cas-9 system:
A
- Cas9 enzyme: cuts DNA at the Target Site
- sgRNA: single guide RNA that brings Cas9 to the target site
- Target Site - DNA to be edited/mutated (your target gene (ytg))
- A PAM site: 5’NGG is required in a specific location at the target site (opposite strand)
25
GTR of sgRNA
These 20 bp are complementary to 20 bp of the target DNA you are trying to mutate
26
sgRNA core
The rest of the sgRNA that binds to the Cas9, the enzyme which cuts the DNA at the target site
27
Does Cas9 have helicase activity?
Yes, as the Cas9 and sgRNA move along the genome, the Cas9 unwinds the DNA, allowing for the sgRNA to see if it can complementarity bind to the DNA
28
What are the two DNA repair pathways that are activated to fix the break?
- NHEJ: Non-homologous end joining pathway
- HR/HDR: Homology directed repair pathway
29
We know that cellular pathways such as NHEJ work to repair the DSB. What is the hope that we have for CRISPR?
- One in a while, the DNA repair occurs incorrectly which leads to small insertions and or/deletions (indels) that can occur
- This will allow us to change our target gene and induce mutations
30
What are indels?
Small insertions or deletions
31
NHEJ actually repairs DSBs correctly ~ 99% of the time. Why then is CRISPR/Cas9 often highly efficient at making indel mutations at a target gene?
- WHen NHEJ repairs the double stranded break correctly, the gRNA + Cas9 system will just rebind to the site and cut it again until there is an error that occurs.
32
Why isn't CRISPR/Cas9 100% effecient?
1) Some gRNAs do not work as well as others in bringing Cas9 to the target gene
2) Most of the time, DSBs are repaired correctly, so on at least a few chromosomes, they will never be repaired incorrectly
33
Protospacer-adjacent motif (PAM site)
- Cas9 will not cut just any DNA with complementary sequence to the gRNA GTR, but only to targets where those 20 bp are immediately followed by a PAM site (5'NGG) on the OPPOSITE strand of the target gene
34
Where does the Cas9 protein cut?
Cas9 cuts the target gene at the phosphodiester bond between the 3rd and 4th nucleotide upstream of the PAM site (1 is started after the N) on BOTH strands
35
PAM is in the target...
gene (NOT in the sgRNA)
36
PAM is on DNA strand the sgRNA...
DOES NOT bind
37
THE GTR AND SGRNA SHOULD HAVE THE SAME SEQUENCE
Therefore, to find the PAM site, just find the strand that has the identical code to the sgRNA and ends up having an NGG region right afterwards
38
*Be careful the DNA can be flipped
1. Find the sequence for the GTR and you will know to find that identical code on the strand that isn't bound to the GTR.
2. Then, identify the 5' end. Start there and go across the sequence that's identical to the GTR, on both sides, to find the NGG site
39
Write out all the crosses that you are doing:
Generation I:
male sgRNA x female Cas9
Generation II:
male Cas9, sgRNA, and ytg x female Dr Sb Hu
Generation III:
individual ytg Sb Hu x female Dr/TM6B, Tb Hu
40
What was the goal of cross 1? Draw it out in full chromosome notation
Male with sgRNA transgene x STock 101 females with act-Cas9 transgene
41
What is act-Cas9 and what does that mean for the progeny?
Act5C-Cas9 is a promoter for actin which is present in every cell (somatic and germline) . By incorporating the Cas9 into this, we will have the Cas9 machinery in every cell of the progeny
42
Actin (act5c)
Promoter drives a high-level transcription of Cas9 in all cells
43
U6:3
Promoter drives a high level of transcription of the sgRNA in all cells
44
What was the goal of cross 2? Draw it out in full chromosome notation
Male with sgRNA transgene x Stock 101 females with nos-Cas9 transgene
*The difference is that these founder animals will not have any mutation in their ytg yet. it will occur in their progeny
45
Nos
Promoter drives a high level transcription of Cas9 in germline cells
46
CRISPR induced germline mutations will be made in ytg in which of the following crosses?
Both Cross 1 and Cross 2
47
What was the goal of cross 3? Draw it out in full chromosome notation
The goal of Cross 3 was to test as a control for the CRISPR/Cas9 system in Crosses 1 and 2 using the ebony gene. If the ebony gene is mutated, which we will see in its phenotype, that can assure us that the act with the CRISPR/Cas9 system is working appropriately
48
What was the goal of cross 4? Draw it out in full chromosome notation
The goal of Cross 4 was to test as a control for the CRISPR/Cas9 system in Crosses 1 and 2 using the ebony gene. If the ebony gene is mutated, which we will see in its phenotype, that can assure us that the nos and with the CRISPR/Cas9 system is working appropriately
49
The ultimate purpose of cross schemes 3 and 4 is to create files with which body color?
Ebony body color
* remember, the name of the mutation is ebony, so if its a WT body color, there is no mutation in the ebony gene. If we CAN induce a mutation in the ebony gene, and therefore make it a mutant, it could have an ebony body color
50
Mosaic
An organism with different genotypes in different cells is called a mosaic
51
In mosaic animals, once a mutation is made, cell division will...
transmit the mutation to a clone of cells
52
The earlier the mutations are made...
The larger the clone can be
53
GSC
Germline stem cells
54
GB
gonialblast
55
Spermatogenesis in Drosophila
Each testis contains 6-9 GSCs that divide through mitosis to give a Gb and another GSC. Each GB undergoes 4 rounds of mitosis to give a cluster of 16 spermatogonial cells. Each spermatogonial cell undergoes meiosis to give 4 spermatids
56
Mutations that are made earlier during gametogenesis...
Will be transmitted to more gametes
57
DNA Polymerase
Key enzyme needed for DNA synthesis (Used for PCR and DNA sequencing)
58
What do you need for PCR?
- DNA Polymerase
- dNTPs
- Primers
- A template strand of DNA
*Adds deoxyribonucleotides to the 3' end of the new chain as it grows
59
PCR
- Uses two primers. These primers target the DNA to be amplified. Note the polarity of the primes (one in the 5' to 3' and the other in the 3' to 5' if I am reading from left to right)
Note that the size of the end PCR product that is made is from the 5' end of the forward primer to the 5' end of the R primer
60
DNA Sequencing (what is the extra thing you need?)
** ddNTPs
- You take your PCR product and melt it to separate the two strands.
- Then, you add a primer so DNA polymerase can start synthesizing new DNA
- A ddNTP can be added (which has a 3' H as opposed to a 3' OH group which stops the synthesis of the DNA strand.
- A ddNTP will be added to each strand, producing tiny fragments that can be lined up and ordered. In automated sanger sequencing (which is what we use), they have a fluorescent label attached to them.
- The tiny fragments will pass through a capillary column that contains a polymer gel that allows for the separation of DNA fragments based on size
- One sorted, the detector will detect the fluorescent wavelength illuminated by each fragmented strand to determine what nucleotide is in that place
CONC OF DDNTPS is 100x less DNTPs
61
Capillary column for gel electrophoresis
The capillary column contains a polymer gel that allows for the separation of DNA fragments based on size.
Here's how the sorting process works:
Injection: The DNA fragments, which are labeled with fluorescent dyes, are loaded into the capillary column.
Electrophoresis: An electric current is applied, causing the negatively charged DNA fragments to migrate through the gel matrix in the capillary. Smaller fragments move faster and travel farther than larger ones.
62
Balancer Chromosomes
- Special chromosomes in Drosophila that cannot recombine with their homologous chromosome (due to multiple inversions)
- Have a dominant mutation (marker) that produces a visible phenotype that identifies individuals bearing the balancer (for example, using the Cy dominant phenotype of curly wings to track the balancer chromosome)
- Have a homozygous lethal mutation (often the same as the dominant visible mutation) so that the Balancer/Balancer homozygotes do not survive
- Genotype Nomenclature: Indicate markers carried on Balancers with a comma:
CyO, Cy. TM6B, Sb Hu
63
Explain the genotype notation of balancers
Before the comma is the actual name of the balancer itself. After the comma is the phenotype associated with having the balancer
64
Why are some Balancer Stocks considered true breeding even though they can have multiple combinations (lecture 4 slide 18)
Because many of the combinations are lethal, so you will only ever get one genotype for the child that survives.
65
How will you determine if you were able to induce a knockout mutation?
1. Use PCR and DNA sequencing to obtain the sequence of potential mutant alleles
2. Compare these sequences to the WT sequence in the UCSC Genome Browser using BLAT to determine if there was a mutation
2. You will evaluate if this is a knockout mutation
66
How to Design Your Primers for PCR and DNA Sequencing:
- Primer 1 should be 500 bp upstream of your CRISPR site that has the DSB included inside it
- Primer 2 should be 500 bp downstream
- Primer 3 should be about 250 bp upstream (used for DNA sequencing)
67
The Central Dogma
DNA --> RNA --> Proteins
68
Transcription Initiation and Elongation
- To start transcription, RNA polymerase must bind to the gene's promoter
- No primer is necessary
- RNA polymerase adds nucleotides to the 3' end of the growing transcript
69
How is the primary transcript processed in EUKARYOTES
- 5' and 3' end modifications
- Splicing also occurs
70
End Modifications
- The 5' end receives a modified guanine nucleotide 5' cap (G cap connected to 3 phosphates of the first nucleotide)
- The 3' end gets a poly-A tail (200 A's at the 3' end)
- The 45' cap adn the 3' poly(A) tail themselves are NOT encoded by sequences in the gene (because they are added on)
71
Splicing Overview
Splicing removes introns from a primary transcript. Splicing can occur in multiple ways leading to different protein products
72
Exons
Sequences in the gene, in the primary transcript, and in the mature mRNA
73
Introns
Sequences in the gene and in the primary transcript, but NOT in the mature mRNA
74
Where does translation start?
Translation does NOT start at the 5'-end of the mature mRNA, but instead at the AUG start codon. The part of the mRNA between its 5' end and the start (AUG) codon is called the 5' Untranslated Region (5'-UTR). THe part of the mRNA between teh stop codon and the 3' end (but not including the poly(A) tail) is called the 3'-Untranslated Region (3'-UTR)
75
What is the start codon?
5' AUG 3' which is the codon for Methionine (M)
76
What are the stop codons?
5' UGA 3', 5' UAG 3', 5' UAA 3' are the 3 stop codons
77
Coding Sequence
The part of the mRNA starting with the codon and ending with the stop codon is called the coding sequence (CDS) or open reading frame (ORF)
78
What do the blue lines of the genome browser represent
The primary transcripts of a gene. The direction of the transcription, exon UTRs (5' and 3'), exon CDS, and introns are annotated in differing thickness.
79
What do the arrows in the genome browser represent?
The arrows shown within the introns indicate the direction of transcription. This is to help you figure out the 5' and 3' ends
80
How do you determine how many alternative splice variants a gene has?
Just count the number of transcripts that pop up on the UCSC genome browser. Each primary transcript is the result of an alternative splice
81
How do you determine how many different proteins a gene has using a gene?
Push together the thicker bars (CDS) and see how many of the thicker bars you need to push together (for example if there is a gap between two CDS, you are counting two CDS to push together)
Then confirm that you don't have any repeats of proteins that you made (to have distinct proteins, you would've had to push together different regions or you just have completely different numbers for CDS that you pushed together). The best way to check this is to push together things and see if they look exactly identical, if they don't look identical, they technically cannot produce the same gene product
82
How to check if a gene has alternative promoters?
First, you must correctly identify which side is the 5' side. If any of the genes does not start in the same part compared to the rest, there are alternative promoters
83
How do you check if your gene has alternative Poly A tail addition sites?
First, you must correctly identify which side is the 3' side. If you see that the 3' does not end uniformly in all your transcripts, you can assume that the poly A tail would also be added in different locations since the genes had different endings
84
Look at slide 32 of lecture 5, is there a second gene in this region of the chromosome? How can you tell?
Kirre is a second gene. You can tell because it is above the gene we are looking at in the same region.
85
What region of kirre does CG3588 overlap with?
CG3588 is completely contained within an intron of kirre
86
Are kirre and CG3588 transcribed in the same or opposite directions?
Both genes are transcribed in the same direction
87
How would you answer the question: How many exons are spliced together to make each of the mature mRNA from these transcripts?
Push together all of the bars and see how many bars you needed to push together to do so (ALL OF THEM)
88
How would you answer the question: The coding region of each of these transcripts is made of sequences derived from how many exons?
Push together all of the thicker bars and count how many of the thicker bars you needed to push to make one solid bar
89
How would you answer the question: The 5’-UTR of each of these transcripts is made of sequences derived from how many exons?
How many of the skinny bars need to be pushed together to make one 5' UTR
*** BE CAREFUL OF SOME OF THE CDS STILL HAVING A BUT OF UTR, COUNT THOSE TOO!
90
How would you answer the question: The 3’-UTR of each of these transcripts is made of sequences derived from how many exons?
How many of the skinny bars need to be pushed together to make one 3' UTR
*** BE CAREFUL OF SOME OF THE CDS STILL HAVING A BUT OF UTR, COUNT THOSE TOO!
91
How do you find which exon the start codon starts in?
Look for the first CDS (thicker bar), the AUG will be in that CDS. Make sure that when you're counting, you're also counting that little bit of 5' UTR that might be included. (this counts as its own exon even though its really tiny sometimes)
92
How to design sgRNA: Understanding the UCSC Genome Browser
When you change the CRISPR output, it will give you bars that are potential sgRNA target regions. This means these are specific segment of DNA where a single guide RNA (sgRNA) could be designed to bind and direct the Cas9 enzyme for targeted genome editing. These regions are typically chosen based on their proximity to a specific gene or mutation of interest, indicating potential sites for CRISPR/Cas9 intervention.
- The thick bars is the 20 bp target sequence that matches the GTR (but this is DNA)
- Arrows in thick bars indicate the orientation
- The thin parts are the PAM sequence
- The color tells predicted efficiency of cuttign (green is good, yellow is medium, red is low, and black is unable to calculate)
93
How to design sgRNA: Selecting the correct sgRNA
- The predicted DSB must be in the earliest (not cutting with ATG) common to all transcripts
*This means that you want the Cas9 to cut pretty early on in the CDS so that every alternatively spliced version can still be mutated. To do this, you want to choose an sgRNA close to the first CDS but not one that interferes with the ATG.
- The predicted DSB must NOT be located within the start codon (ATG) itself (internal ATG codons are not relevant)
- The target site should have a high (green) predicted cleavage score
- There should be zero off target sites in the 0 and 1 mismatch categories
94
If you see ATG in the DNA, what strand are you working with? What strand of DNA does the sgRNA bind to?
- The coding strand.
- The sgRNA is identical to the coding strand so it binds to the template strand
95
So... which sgRNA should you choose?
You want to target a region within the coding region of your target gene as close as possible to the start codon (but NOT in the start codon)
*It helps to write out what the PAM sequence is to know that you would be cutting up between the 3rd and 3th nucleotide
96
Make sure you choose the CDS that is the first one in common with all the splices - even if its the last one!
97
How does DNA Agarose Gel Electrophoresis work?
You load your sample into wells that are closer to the negative electrode. Once you turn on your power source and create an electrical current, the DNA (which is negatively charged) will move away from the negative electrode and towards the positive electrode.
DNA fragments that are smaller in size will move towards the bottom rather quickly while those that are larger will move slowly
You should always have a ladder (different fragments of DNA with known sizes) to reference your DNA to
98
How to determine the mRNA-like strand vs the template strand
If the arrows are moving from right to left: Template
If the arrows are moving from left to right: Coding
*You can also tell because when you zoom into the DNA to see what the nucleotides are at the Start (Green), if it is CAT, it must be the template because it is the reverse complement. If it says ATG, it is the coding strand (or mRNA like strand)
*Go to voice note for explanation
99
How to determine the open reading frame
To determine the open reading frame, you want to select only the options of the coding strand technically. Or you only want to work with finding the different frames of the CODING STRAND because if is moving in the same direction as the mRNA,
Find the ORF that doesn't have a stop codon
100
When do you know if you have a knockout mutation?
If you have a deletion or insertion of 1. you will have a frameshift. If its very early on, its a knockout
If you have a multiple of 3, it might or might[[[ not be a mutation
