Gene technology 3-8 Flashcards
What’s exon trapping?
You can identify if genetic material contains any exons or if they’re solely intronic.
- Insert genetic material in a splicing vector containing exon-intron-exon material.
- Let the vector replicate in vivo. If the inserted genetic material hosts an exon, the resulting mRNA after splicing will be larger than the control. If there’s only intronic DNA in the material entered, the mRNA will be of the same size.
What’s a primer extension assay?
You can find the transcriptional start site (5’) using radio-labeled primers. The primers need to be complementary near the 3’ end of the transcript and it’s going to facilitate RTs elongation of cDNA.
In order to fin the exact transcriptional start site, you’ll have to run a sanger seq.
What’s EMSA?
You find if protein and DNA interact by assaying for shifts when running a gel. You can identify the protein (as long as its not novel), by adding antibodies - this is referred to as supershift EMSA.
What’s a DNA footprint analysis?
You assay for DNA - protein interactions within a sequence of interest.
- Amplify the desired fragment.
- Mix protein with DNA.
- Add enzymes that cleave randomly. The reaction should be long enough for avg 1 cleavage / DNA strand.
- Run gel. The unbound DNA will be visible in a ladder distribution. Any breaks in the ladder indicate that the DNA was protected by the protein of interest. It’s much like ribosome footprinting or the histone-equivalent.
What’s a yeast one hybrid system?
The method is based on a prey (protein) and bait (DNA) plasmid. The prey sequence is conjugated to an activating domain which recruits the transcriptional machinery if prey-bait interaction occurs. Downstream you’ll find a reporter gene.
Worklow
1. Generate and transfect bait construct containing the DNA sequence which will be checked for protein binding + selection gene. The construct will integrate into the yeast genome.
- Generate prey construct + AD protein as a fusion protein. Transect into the same yeast cells as the bait construct.
- If prey-bait interaction occurs, the reporter gene is transcribed.
What’s a yeast two-hybrid system?
The premise is the same as for yeast 1 hybrid assay. The difference is that Y2H assays for protein-protein interactions. It doe this by segregating a transcription factor into two proteins which must interact in order for the transcription factor to become active and transcribe a downstream reporter gene.
The assay is in essence a mimicry of a transcription factor only if the two splits (bait and prey) are present.
What’s shotgun sequencing?
Shotgun sequencing refers to the way DNA is fragmented before sequencing it. The term refers to a random pattern of fragments occurring w/ various overlaps.
Describe illumina seq.
- There are oligonucleotides anchored to the Illumina plate which are complementary to adapter a or b. The fragments (w/ adapters) bind to their respective oligos on the flow cell.
- PCR reaction synthesizes the complementary strand to the fragment which is attached through its adapter to the flow cell.
- The dsDNA is denatured, and the unbound fragment (the newly synthesized fragment) is washed away.
- The strand attached to the flow cell bends over due to heat cycling, and the second adapter binds to an anchored oligo. PCR reaction occurs again, either one-ended or pair-ended, leading to one-end or pair-end reads. pair-end reads are beneficial.
- The dsDNA (in the bridge) is denatured, the flow cell is washed, then the process is repeated.
- Sequencing primers are added (for the adapters). Fluorescently labeled nucleotides are added (all have different fluorescences). For every nucleotide, the reaction is exposed to exciting wavelengths for the different fluorophores.
Describe SMRT seq
- Target nucleic acid (RNA or DNA) is conjugated with circulating adapters.
- One circularized nucleic acid molecule is added to each well on the SMRT plate with polymerase (attached to the well), dNTPs, Mg2+…
- dNTPs emit different fluorescences which are read from under the plate.
A: To what degree must a single nucleotide polymorphism be present in a population for it to be an SNP?
B: What’s a haplotype block?
C: How often does recombination occur in meiosis?
D: How can you assay for SNPs (2 methods)?
A: >1%
B: Inherited blocks of genetic code inherited from meiosis.
C: 16-29 times.
D: GWAS, SNP arrays
What’s AMRS-PCR (Amplification refractory mutation system?)
It’s a PCR method which requires perfect primer complementarity. It’s good for assaying known SNPs, as the resulting amplicons must contain it for amplificaiton.
(See if 100% correct)
Describe what a high resolution melting (HRM) analysis is.
HRM is an analysis of amplicons where you suspect an SNP to reside. You compare the melting curve of your suspected SNP w/ a WT DNA strain. AT and CG melt differently.
What’s iPLEX?
- Amplify a region in which you suspect you have an SNP.
- Re-amplify the amplicon using mass-modified ddNTPs. The chain will terminate after the incorporation of the nucleotide and each amplicon will have different sizes so that you can assay the incorporated nucleotide’s identity on a gel.
What’s array comparative genomic hybridisation (aCGH)?
an aCGH is a chromosome-wide array of WT fragments compared to test samples. You can assay for known SNPs by having them represented by the oligos on the plate.
