gn735_Final01

• KASP
• denaturing HPLC
• TILLING
• ASO
• DASH
• Flurogenic dye based method: TaqMan, Molecular beacons
• Dye-labeled oligonucleotide ligation assay
• FRET

• Kommpetitive Allele Specific PCR
• >99.8% accurate
• phenotype polymorphic sites. Doesn't get them all, but can type many individuals at once
• supports low and medium thru put studies

• 1. three ingredients in the assay mix: test DNA with the SNP of interest, KASP assay mix (contains two different, allele-specific competing forward primers and one reverse primer), KASP Master mix (containing FRET cassette +Taq polymerase)
• 2. Template is denatured and PCR begins. Only one of the allele specific primers match target SNP, and the common reverse primer amplifies target region
• 3. Complement of allele-specific tail generated by PCR.
• 4. Signal generation: the fluor labeled part of the FRET cassette is complementary to the new tail sequence and binds, releasing the quencher and generating a signal

• Variant detector array
• looks for DNA sequences that differ by SNP
• Examines a large number of SNPs per assay
• very expensive per sample, b/c tests four nucleotide for each base to see which is a match
• "high technology"

• We take our reference genome and design our array from it.
• Each cell interrogates one nucleotide to give a lot of redundancy
• Strong hybridization that matches the reference genome exactly
• weak hybridization in the surrounding area if the SNP is present

the most common way to compare levels of RNA in a sample

qPCR uses fluorescent dyes (e.g. SYBR) or TaqMan probes for a rapid and accurate, real-time quantification of RNA and is expressed as the number of gene copies or target organism (genome) in a sample

• fluorescent probe attached to the 5' end
• Taq polymerase cleaves a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection
• As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection

• capture probe
• reporter probe
• direct counting of the gene sequence you want to interrogate

• Take a tissue sample you want to analyze
• hybridize with probes
• Now you can see the transcript of several genes at the same time, with their spatial information

• Start with your sample, create an emulsion that separates your sample so that only a few molecules are present in each partition
• each 'bead' is about one nano liter
• Each bead undergoes PCR with TaqMan assays, allowing for quantity of molecules in a droplet to be analyzed
• After several cycles (30?) the droplets are separated and the quantity of the molecule is recorded
• The information follows a Poisson distribution, allowing for calculation of the starting material in the distribution

get information about spatial/temporal expression information

illustrates where a gene is transcribed into RNA within a cell

illustrates where the RNA is translated into a protein within a cell

• Select the cDNA to print (either a uni-gene set or random clones from a normalized cDNA library)
• goal is to determine differential expression of genes in different conditions

• 25nt oligos (minimum length for hybridization)
• required four masks per nt in the oligos you wanted to put on the array

• could make oligos longer than the affymetrix arrays
• used mirrors that could be controlled with a program

• Serial analysis of gene expression
• first attempt to do high thru-put sequencing without next gen sequencing

• PacBio
• Oxford NanoPore

• A standard curve helps you accurately asses the actual quantity of DNA/plasmid in your experimental sample.
• The standard curve is constructed using several different known concentrations of DNA containing your target sequences.
• These are plotted and a regression model is made
• From this regression, we can accurately predict concentration of our sample

• a method to determine identify of a nucleotide at a specific position along a nucleic acid.
• used to identify SNPs
• A oligonucleotide primer hybridizes to a complementary region along the strand
• DNA Polymerase extends the primers until it reaches a specific position you want to interrogate for the SNP
• A labeled (for visualization) terminator is incorporated at the desired site, and indicates the complementary bp to the nucleotide present

• Matrix assisted laser Desorption Ionization- Time of Flight
• Laser hits the matrix and breaks up/sends whatever you want to analyses into the gas phase to be analyzed by mass spectroscopy
• time of flight aspect also help identify what protein it might be

• used to measure the transcriptional activity of genes in cells
• Isolate the nuclei and purify
• incubate the nuclei with a radioactive labeled nucleotides which will be incorporated into transcribed RNA
• Use a probe to hybridize to your gene/RNA transcript of interest and measure the amount of radioactivity
• How much RNA for a particular gene is being made?

• light is detected when a dNTP is added one at a time
• genotyping analyzes many samples, but only for one individual

• High technology method for genotyping
• hybrid between KASP and extension systems
• used to simultaneously analyze hundreds of thousands of genetic variants in a single experiment
• GoldenGate assay allows for genotyping directly on genomic data, without the need for PCR

• use restriction enzymes to digest your DNA
• ligate adapters with barcodes specific to each well for identification
• PCR amplify the digested products
• sort by sample size to further reduce number of fragments to sequence (like b/w 100-400 nt)
• Sequence your remaining fragments
• Relatively cheap and can investigate many SNPs and individuals at once

• examines presence and abundance RNA levels (historical and no longer often used)
• Use a control gene to accurately measure RNA difference for correct "fold-increase" information