Methods 3 Flashcards
What sequencing technology will you use to confirm that the mutant maize seed stock you got from another lab harbors the mutation of interest? What will you need to do to sequence?
Sanger
You know what you would expect to see, you would design primers up and downstream from the mutation (within 70 - 100 or 500 bp away), pcr amplify, sequence with sanger because you only have to sequence that particular amplified strand (not the whole genome), also sanger has the advantage over short-read sequencing technologies (like Illumina) in that it can produce DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%.
What sequencing technology will you choose to identify mutations in a patient’s tumor? What steps will be involved?
PacBio and Illumina combo
As there are many possible mutants that could be present you need to do full genome sequencing (next gen/2nd gen). PacBio (SMRT) provides very long reads (10-25kb) so you can use it to id chromosomal rearrangements, but it is low fidelity (85-95%) and sample throughput is lower than Illumina sequencing platforms. Illumina has short reads but are more accurate and is very fast as sequences in parallel, you can fit the sequence back together using pacbio reads too.
What sequencing technology will you select to identify genetic differences between two different populations of mice of a species for which a good-quality reference genome is available? What steps will be required?
Illumina
fragment, add adaptors, end repair, DNA amp, sequence.
if no reference genome is available use pacbio to make it easier to assemble contiguos sequences
Why do you think Illumina sequencing alone is typically NOT enough to generate a reference genome of a new species?
illumina gives sequence quality, pacbio gives larger arrangements
illumina is high coverage and confirms sequences from pacbio
often repetitive regions are hard to sequence/are ambiguous (so pacbio is good with repetitive sequences)
why does sanger require two PCR reactions?
one for the forward primer and another for the reverse primer
what 2 DNA sequencing technologies are good for repetetive reads
pacbio and oxford nanopore
________ sequencing is needed to build a reference genome
long-read (pacbio)
explain oxford nanopore
A protein unzips the DNA helix into two strands
A single strand of DNA is threaded through a tiny protein pore in a synthetic membrane
An electric current flows through the pore
Different DNA bases disrupt the current in different ways
The machine measures the current and interprets the sequence (aka “squiggles”)
Drawbacks: higher error rates and lower throughput
Pluses: portability and ease of use; can also sequence RNA
explain the adaptors used in oxford nanopore
DNA fragments are ligated to two adapters: a Y-shaped “leader” adapter and a stem-loop “hairpin” adapter
Adapters direct the DNA fragments to the pores by binding to tethering oligos with affinity for the polymer membrane
Adapters are preloaded with ‘motor proteins’ that enable the threading of adapted 5’ end through the pore
how many bp reads does oxford nanopore do?
A MinION flowcell contains 512 channels (4 pores each) that thread 512 DNA molecules at once
10-100kb and 100-300kb sequencing modes are available (with a 4Mb read being the record)
what is a reporter gene
Reporters are genes that are used in molecular biology to monitor expression of genes of interest (GOI)
where are reporters fused
Reporters are fused to promoters, gene fragments or entire GOI and their activity is assayed/analyzed
what does luciferase emit and what DNA sequencing is it method is it used in
photons
454 pyrosequencing
example of two reporter genes that convert colorless substrate to blue and name the substrates
gus (gluc) and lacz (x-gal)
explain a transcriptional reporter gene fusion
the reporter gene is fused directly next to the promotor
tells you where the gene is transcribed, GFP only be expressed in tissues at time of transcription
explain a translational reporter gene fusion
the reporter gene is fused to the open reading frame so GFP is incorporated into the protein, tells you only when a protein is translated and tells you where that protein resides
How to test if a reporter fusion protein has perturbed function?
knockout then expression of fusion protein to see if function is restored
terminal fusions are common but if function is perturbed must go inside the structure, algorithms can be used to find where.
What type of a reporter (transcriptional or translational) will you use to study:
Tissue-specific pattern of expression of a gene of interest
do both and compare to know if expression and translation occur in the same tissue/place
What type of a reporter (transcriptional or translational) will you use to study:
Subcellular localization of the protein of interest
translation (you want to know where the protein goes)
What type of a reporter (transcriptional or translational) will you use to study:
“Inducibility” by heat shock of the gene of interest?
either depending on if you’re interested in studying it at an expression level or translational level
What type of a reporter (transcriptional or translational) will you use to study:
Protein movement
you need both to understand if the protein is moving from where it is transcribed
What type of a reporter (transcriptional or translational) will you use to study:
Regulation of translation by nutrient deprivation
both because nutrient deprivation could be impacting both
what is northern blot analysis useful for?
to study expression (levels and patterns) of the genes of interest by blotting RNA and hybridizing with radio/fluorescently labeled DNA probes, a housekeeping gene is used for normalization
what is in situ hybridization
A hybridization-based method that enables detection and localization of endogenous mRNAs at cellular level
Gene-specific probes (cDNA or, more commonly, cRNA) are labeled (radioactively (A), fluorescently (B) or using base modifications such as digoxygenin (C)) and hybridized to fixed permeabilized tissue sections or entire small organs/organisms
Probe hybridization is detected using autoradiography (A), fluorescence microscopy (B) or immunohistochemistry (C)
explain DIG method of in situ hybridization
The digoxygenin (DIG) can be synthetically linked to the C-5 position of uridine nucleotides
The DIG-labeled nucleotides are incorporated into gene-specific RNA probes
Hybridized DIG-labeled probes are then detected with high-affinity anti-DIG antibodies conjugated to an enzyme (e.g., alkaline phosphatase) or a fluorescent dye
explain DNA Fluorescence In Situ Hybridization (FISH)
(the colorful pictures of chromosomes)
(occasionally used for RNA)
cytogenetic technique for the detection and localization of specific DNA sequences on chromosomes
FISH uses fluorescent DNA probes that bind to specific chromosomal regions with high degree of sequence similarity to the probe
used to investigate chromosomal abnormalities
