Technology Flashcards
similarities of DNA and RNA microarrays
both end with DNA microarray
DNA microarray
do for entire genome
RNA microarray
only target those genes
purify RNA
make cDNA
aCGH (array comparative genomic hybridization)
saw huge differences and compared reference to genome to another genome
Genome wide technologies
DNA/RNA microarrays
aCGH
NGS (next generation sequencing)
applications of DNA microarrays
- gene transcription measurement
- SNP/CNV
- chromatin immune precipitation
- clinical diagnostics
uses glass surface with probes with a specific nucleotide sequence to anneal to DNA
DNA microarrays
hybridization based technology
denature so cDNA can find probe and bid firm
wash DNA not attached
how many unique features do DNA microarrays have?
2.1-3 million
uses DNA with chromophore
DNA microarrays
how many different probes are there for each gene in DNA microarrays?
22
southern blot
dna
northern blot
rna
western blot
protein
most common
two microarray platforms
spotted arrays
affymetrix
uses 2 colors and glass
spotted arrays
1 or more color to compare 2 samples against each other
affymetrix
which is more common?
spotted array or affymetrix
affymetrix
what do spotted arrays contain?
oligonucleotides 50-70mer
What are samples labeled with in spotted arrays?
CY5 (red)
CY3 (green)
samples loaded and mixed and hybridized simultaneously to a single slide
spotted array
oligonucleotides to detect genes
anneal to certain area of gene
multiple areas
what percent of genome is expressed in oligonucleotides?
2%
what is RNA used to measure?
gene transcription
what is DNA used to measure?
CNV or SNP difference between genomes
what do aCGH measure?
congenital abnormalities
cancer
how many oligonucleotides are used in aCGH?
> 1 probe per kb genome wide in human
what is the length of oligonucleotides used in aCGH?
25-100 bp
t/s arrays function well for high copy number sequences
false
is the control or cancer labeled green?
control
is the control or cancer labeled red?
cancer
what color is shown if cancer and control are expressed in both?
yellow
what length do affymetrix use of oligo?
25 mer length
how many different olives are there for each gene in affymetrix?
22
single sample goes on a single array located in cartridge
affymetrix
illumina SNP genotyping
know difference between normal or WT allele
SNP extension
what is exon sequencing?
next generation sequencing
sanger sequencing
DNA sequencing
capillary dna sequencers
whole genome project
next generation DNA sequencing
multiple applications
the reality of whole genome sequencing at a reasonable cost
have a piece of DNA you want to sequence and need multiple copies of this part of DNA
sanger sequencing
separation b electrophoresis, fluorescent tags
sanger sequencing
what terminate the reaction in sanger sequencing
dNTPs
ddNTPs
sanger sequencing outcome
dozens of gels
label with p52
deep sequencing outcome
one run
5 days
How how does it take to analyze the DNA in whole genome sequencing?
2 months/2 days
what company was formed for NGS?
454
sequencing in 454 company
emulsion PCR
pyrosequencing
single molecule PCR in microsphere compartments
emulsion PCR
chemiluminescent detection of pyrophosphate
pyrosequencing
use sedimentation and fragmentation with oil in water emulsion
emulsion pcr
what do you add in emulsion pcr
micro beads
