Midterm 2 Flashcards
What are the steps to sequence a genome? (6 steps)
- Fragment(cut up) the genome with restriction enzymes(specific places) or sonification(random)
- Clone DNA fragments into a plasmid-vector backbone
- Transform recombinant DNA molecules into E.coli and plate to get colonies
- Purify recombinant DNA molecules
- Perform paired-end sequencing
- Assemble reads into contigs and use paired-end information to assemble contigs into scaffolds
Deletion
A single chromosome has two double-stranded breaks. The region between the breaks is lost and the remaining parts join together via NHEJ
Inversion
A single chomosome has two double stranded breaks. The region between the breaks is stitched back into place but only after rotating 180 degrees
Deletion/Duplication
Two homologous chromosomes/sister chromatids are broken at different locations. Can result in deletion or duplication depending on which pieces are put back together
Translocation
Breakage of two non-homologous chromosomes results in reciprocal transmission
transposable elements
DNA sequences that can move from one location in the genome to another
Enhancer
A DNA sequence that regulates gene expression, is tissue-specific, and can be upstream/downstream
What do reporter assays do?
Test enhancer activity
What three things do you need for a reporter assay?
- Putative Enhancer: you test its activity
- Minimal Promoter: for polymerase to bind and transcription to happen
- Reporter gene: to screen for activity of the putative enhancer
Transcription factors
proteins that bind at specific sequences(enhancers/promoters) to regulate(activate/repress) gene expression
ATAC-Seq Steps
- Tagmentation using Tn5 transposase (cut accessible chromatin and simultaneously fragment and tag accessible regions)
- Use paired-end sequencing
What is ATAC-Seq used for?
Determines chromatin accessibility
What is Chip-Seq used for?
Used to analyze protein interactions with DNA. Identifies the binding sites of DNA-associated proteins (transcription factors + enhancer/promoter interactions)
What are the 3 components of Chip-Seq?
- Formaldehyde: crosslink - sticks protein onto DNA
- Sonication: for fragmentation
- Antibody: for co-immunoprecipitation; labels protein of interest
What are the 4 steps of Chip-Seq
- Crosslink the DNA and proteins using formaldehyde
- Fragment the DNA using sonication
- Co-immunoprecipitate the transcription factor bound DNA using a specific antibody
- Purify the DNA and sequence
What’s the difference between forward and reverse genetic screens?
Forward begins with phenotype of interest. Reverse begins with a gene of interest.
List the 4 steps of CRISPR/Cas9 in bacteria
- Transcription of CRISPR locus
- Translation of Cas9 mRNA into protein and processing of pre-crRNA into shorter crRNAs (tracrRNA base pairs with repeat sequences and RNase III cuts there)
- Mature crRNAs each contain a spacer and repeat sequences
- When an invading virus injects its DNA into the bacteria, the matching crRNA tracrRNA and Cas9 corporate to cleave and degrade viral genome
- 3’ end of crRNA pairs with tracrRNA to form hairpin loop that binds Cas9
- 5’ end of crRNA base pairs with target DNA in the phage DNA and Cas9 recognize the PAM sequence
List 4 steps of CRISPR/Cas9 technology
- Cas9 recognizes PAM site and the guide RNA anneals to target sequence
- Cas9 cleaves DNA - double stranded breaks
3a. If no introduction of exogenous plasmid/construct(DNA template) -> NHEJ -> knock out
3b. If introduction of exogenous plasmid/construct -> HDR -> knock-in
What is the GAL4/UAS system used for?
The system is used for expression of a specific gene in a specific tissue
What is GAL4
transcription factor that binds UAS sequence; typically under the control of a tissue-specific enhancer
What is UAS
A simple enhancer sequence for gene X; has many binding sites for GAL4
How does the GAL4/UAS system work?
The GAL4 transcription factor is only produced in specific tissue. Thus, the GAL4 binds UAS and facilitates transcription of whatever is downstream of the UAS sequence(gene X). You need both GAL4 and UAS to facilitate transcription of gene X.
In situ hybridization
Utilizes a single-stranded, sequence-specific RNA probe to visualize mRNA of interest
Chromogenic in situ
Probes are conjugated to enzymes that, upon addition of a substrate, will produce a colored precipitate at location of mRNA of interest.
Fluorescent in situ
Same idea as chromogenic in situ but instead mRNA is visualized by fluorescence as opposed to a colored precipitate
Immunolocalization
uses an antibody to visualize a protein of interest
What are the 3 types of quantitative traits?
- Continuous: traits that vary continuously(weight, height, etc.)
- Meristic: measured in whole numbers (animal liter size, # of flower petals, etc.)
- Threshold traits: measured by presence or absence (susceptibility to disease)
What is the goal of QTC?
The goal is to identify specific regions on chromosomes that are asspciated with variations in a complex quantitative trait
What are the 7 steps of QTL mapping?
- Identify the quantitative trait you want to study
- Obtain two individuals that vary greatly in the trait you want to study
- Cross two parental tomatoes together to generate F1 generation
- Cross F1 with another F1 to generate F2 generation
- Score phenotypes of F2 generation (measure their weights and put them into groups based on weight(
- Genotype each F2 individual for a panel of known SNPs(molecular markers) throughout the genome
- Perform statistical tests to determine if there are certain SNPs that show linkage with the weight of the tomato
List the 3 steps of GWAS
- Collect DNA from many cases(disease status) and controls(unaffected)
- Genotype everyone for tag SNPs throughout the genome
- Test for association of each SNP genotype with disease status
Describe the process of paired-end sequencing (two steps)
- The DNA fragment of interest that you want to sequence is inserted into a vector backbone with a known sequence
- primers are designed to the vector backbone. Primers sequence ~150bp from either end of the DNA fragment
What does paired-end sequencing tell us?
With paired-end sequencing we know that Read1 (coming from one end of DNA fragment) and Read2 (coming from other end) are coming from the same piece of DNA. This is helpful for assembling contigs into scaffolds
What can happen during homologous recombination?
A double stranded DNA strand breaks, and a section of a DNA template(plasmid) gets integrated into the broken double stranded DNA. Homologous recombination between repetitive sequences (eg. transposons) can lead to
duplications when repetitive sequences are on the same chromosome in the same
orientation.
What is NHEJ(Non-homologous end joining)?
Paste back together the double-stranded DNA after a breakage
How do reverse genetic screens work?
You do a reverse genetic screen when you do know which genes might be responsible for the phenomenon you want to study. You directly disrupt those genes and see if they result in the phenotype of interest.
What are complementation assays used for?
Used to determine if two or more mutations are part of a single gene or of different genes.
