3. DNA analysis Flashcards
(17 cards)
transcriptome
all transcripts produced by a cell, tissue or organism
GAPDH
common reference gene, doesn’t change expression much with environmental stimuli
gene analysis tools
- sequencing
- restriction enzyme digest
- electrophoresis
- PCR
- cloning
- gene and primer synthesis
- CRISPR Cas
comparitive sequence analysis
- can check the functional importance of amino acids
- conserved across species usually means more important
searching seq databases
- find particular genes in particular species
- possible gene functions
- possible significance of variants
identifying genetic markers of disorder
- look for STR identifiers common between only infected individuals
- consult literature
- identify candidate genes then PCR and sequence
- compare to reference genome
identifying candidate genes
- identify all DNA changes
- remove neutral changes
- remove changes that are unlikely and/or have the wrong inheritance pattern
- final contestants
PCR key features
- allow for exponential increase in DNA fragments
- Primers determine what is amplified
- temperature is the key to specificity
PCR temperatures
Denaturation: 95 Degrees Celsius
Annealing: 50-67 Degrees Celsius
Extension: 67-72 Degrees Celsius
Dideoxynucleotide
Molecular mimic of deoxynucleotide, but cannot extend DNA molecules
- used for Sanger sequencing
Sanger sequencing
Sequencing by synthesis.
- reaction has DNA fragment, ddNTPs each linked to a different fluorescent dye, dNTPS
- DNA molecules are separated by size and a complementary sequence can be seen
Next-gen sequencing stages
- DNA library preparation
- Hybridisation to flowcell
- one nucleotide detected per cycle
- computer generates sequence
Next-gen-seq library preparation
- shear DNA
- Ligate adapter = Primer
- PCR amplification
Next-gen-seq Hybridisation to flowcell
- flowcell contain adapators use to generate DNA clusters
- clusters allow for stronger fluorescent light
- one human genome = ~2 flowcells
Next-gen-seq nucleotide detection
- each dNTP has a terminator and fluoro tag
- after detection the terminator and tag are cleaved
- slows process but increases accuracy
RNA-seq
very similar to DNA seq
- sequences the transcriptome
- RNA must first be converted to cDNA
- work out changes in expression of genes and alternative splicing
RT-qPCR vs RNA seq
RT-qPCR: measures the expression level of a known gene
RNA-seq: measure the expression level of all genes in a sample
