what is the central dogma of biology?
DNA (long term storage) is transcribed into RNA (that turns into proteins)
what is gene expression?
- gene expression is the process where the information encoded in a gene is turned into a function.
- mostly occurs via the transcription of RNA molecules that code for proteins
list ways to measure mRNA expression (3)
Quantitative rtPCR (main way)
Microarrays (becoming old fashioned)
RNA-Sequencing
what information can be analysed from mRNA expression?
- Measure and compare RNA expression
- Estimate environmental or genetic effects on phenotype
- Find differences in gene expression that could explain differences in phenotypes
requirements for PCR/rtqPCR
- Template DNA
- H2O
- Precise thermal cycling
- Heat stable polymerase
- Nucleotides
- Oligonucleotides (primers)
PCR steps
- Denaturation - take template DNA and heat to 90* for 1 min
- Annealing - lower temp to 54* and wait 45 seconds. The primers will bind to DNA, complementary to their seq.
- Extension - Polymerase extends DNA using nucleotides in solution. 2 mins at 72*
what makes a “good” primer (oligonucleotide)?
- Lacks secondary priming sites – will only bind to one area in genome
- A melting temp (TM) between 52* and 65*
- Absence of dimerization capability - don’t want primers to bind to themselves. Eg, we wouldn’t have a primer that is AAAA and another that is TTTT.
- Absence of significant hairpin formation – primer folding back on itself. >3 bp
- Low specific binding at 3’ end
describe the “uniqueness” of a primer.
There shall be one and only one target site in the template DNA where the primer binds
Primer sequence shall be unique in the template
There shall be no annealing site in possible contaminant sources, such as human, rat, mouse, etc.
BLAST search against corresponding genome
describe the importance of the length of the primer.
Primer length has effects on uniqueness and melting/annealing temperature.
the longer the primer, the more chance it’s unique
the longer the primer, the higher annealing temperature.
Generally speaking, the length of primer has to be at least 15 bases to ensure uniqueness. Usually, we pick primers of 17-28 bases long.
describe base composition of the primer.
Base composition affects hybridization specificity and annealing temperature.
Random base composition is preferred. Avoid long (A+T) and (G+C) rich region if possible.
Usually, average (G+C) content around 50-60% will give us the right melting/annealing temperature for ordinary PCR reactions
list the 3 types of secondary structures to avoid during PCR.
- hairpin (primer folds back and binds to itself)
- self-dimer (2 dimers binding together)
- dimer (forward primers bind to reverse)
describe the importance of primer pair matching.
Primers work in pairs: forward and reverse
Critical feature is annealing temp – must be compatible for both. Max difference is 3*C.
q rtPCR steps.
- Design primers for your gene of interest (gene X)
- Design primers for a house keeping (HK) gene
- Perform real time qPCR reaction
—> Determine at which cycle the intensity crosses the threshold - Relative expression of gene X versus a HK gene
ADV and DISADV for qrtPCR
– small number of genes (targeted 1/2/3 genes)
+ large number of samples (5 or 500 would be similar price)
what are housekeeping genes?
Housekeeping genes encode proteins that are usually essential for the maintenance of cellular function and often remains constant under most experimental conditions.
eg., transcription factors, GAPDH, Ubiquitin C (UBC).
what is rt qPCR?
Quantitative reverse transcription polymerase chain reaction.
- real time PCR using fluorescent probes, that bind to DNA, for analysis
ADV of rt qPCR?
- accurate
- reliable
-extremely sensitive
what is the difference between biological and technical replicates??
biological replicates are being measured as a sample and technical replicates are a repeat of those samples with the only purpose being a measurement of precision.
- we DO NOT want a lot of variance
mathematical steps for quantitative rtPCR. (5)
- Compute mean of technical replicates.
FIND MEAN HK AND MEAN GENE X. - Find the delta Ct between these values.
GENE X mean - HK mean = delta Ct - Of the previous values, find the mean of the delta Ct for the control samples.
- Find the delta delta Ct.
SUBTRACT THE MEAN DELTA CT FROM THE DELTA CT. - Compute the 2^-delta detla Ct.
- Interpret results.
what are delta Ct values reliable?
when the value is >0.3 away from the median (middle) value of the other samples.
what would we do if delta Ct isn’t reliable?
- if CONTROL GROUP is unreliable, us the geometric mean of the control group. This mean is less sensitive to an outlier.
- when there’s a SINGLE OUTLIER, remove offending bio. replicate.
- or REDO whole experiment.
what is a micro array?
A micro array is a collection of microscopic DNA spots attached to a solid surface - usually a glass plate 9x12cm
what is in a micro array spot?
Each spot contains ~ 10−12 moles of a specific DNA sequence, known as probes
- Probes are also called reporters or oligos
how is the DNA targeted in micro arrays?
the DNA (which can catch other pieces of DNA) are targeted with
