Module 7: Microbial Genomics (Transcriptome + Proteome Methods) Flashcards
Gene expression CANNOT be determined from ____________
Why?
Gene expression CANNOT be determined from DNA sequence alone
Because regulation processes of genes do not alter the DNA sequence itself so its sequence can only tell us what genes may be present but not which ones are on/off
What DOES whole genome sequencing tell us? (2)
What does it NOT tell us?
DOES tell us:
1) Proteins potentially present + potential functions of them
2) Evolutionary relationships between organisms
DOES NOT tell us:
–> Expression of genes (what proteins are ACTUALLY present at a given time in a cell)
Analyzing expression patterns of organisms allows us to do what?
Allows us to better understand how microorganisms FUNCTION + ADAPT under different conditions!
Transcriptome
Set of transcripts encoded by each of the genes within a genome
(Collection of transcribed mRNAs in a cell)
For analysis of the transcriptome, what fundamental technique do all methods rely upon?
Nucleic Acid Hybridization (complementary binding)
Nucleic Acid Hybridization
A technique in which ssDNA + RNA molecules bind to their complementary sequences (enabling detection + identification of specific sequences)
What are the methods for transcriptome analysis (that we learned about)?
1) Northern Blotting
2) Microarrays
3) RNA-seq
4) Q-PCR
Northern Blotting
(A modified version of southern blotting)
A technique for the detection of specific RNA sequences by hybridization (using labeled probes w/ complementary sequences)
Southern vs Northern Blotting
Southern = Detects specific DNA sequences by hybridizations (came 1st)
Northern = Detects specific RNA sequences by hybridization (a derivation of the southern method)
A southern blot provides info about what in a genome?
About what genes are present/absent in a genome
What is the process of a northern blot?
1) Extract TOTAL RNA from cells
2) Total RNA separated via gel electrophoresis
3) Transfer separated RNA bands from gel to NC or nylon membrane (make sandwhich)
4) Link the transferred RNA molecules to the membrane (bind them to the membrane) –> Using UV light
5) Incubate membrane with labeled probe/s
6) WASH membrane
7) Detect presence + strength of probe signal
Northern Blot:
What are the bands of the RNA gel electrophoresis?
Upper prominent bands = rRNA
–> Because rRNA is most abundant RNA in cells!
Lower smear below rRNA bands = predominantly mRNA and some tRNA
Northern Blot:
What is the transfer step?
The transfer of the separated out RNA sample from the delicate gel to the more sturdy nitrocellulose or nylon membrane
Northern Blot:
What are the components of the northern blot sandwich?
Bottom (Dish):
1) Transfer buffer
2) Sponge
3) GEL
4) MEMBRANE
5) Paper towel stack
6) Glass plate
7) 0.5 kg weight
Northern Blot:
Why is the transfer step needed?
Because gels are really fragile!
To prevent breakage and sample loss, we move the contents to a more supportive structure (nylon or nitrocellulose membrane)
Northern Blot:
What is the linking step? (How is it done? WHy?)
Linking of the transferred RNA to the membrane
How = Through UV light treatment!
Why = To immbolize the RNA on the membrane and prevents loss of sample RNA during next steps
Northern Blot:
What are the potential labeled probes that can be used?
1) Radiolabeled probes
2) Chemiluminescent probes
Northern Blot:
What are the probes utilized? What are the made of?
Probes = ssDNA molecules designed to hybridize with speficic RNA strand of interest
(pair with via complementation
Northern Blot:
Hybridization between probe + RNA will only occur if…
If there is enough sequence similarity between the probe and the RNA of interest!
Northern Blot:
Why is the washing step important?
Washing occurs after probing to remove any unbound probe
== Ensures that there is no signal coming from any band in which hybridization did NOT occur
–> Ensuring only the labeled probe remains if it’s BOUND to the target RNA of interest!
Northern Blot:
If at the washing step no RNA of interest is present, what happens?
ALL of the probe added to the mixture is unhybridized and is washed away!
–> None of it bound to the RNA on the membrane
Northern Blot:
Detection of hybridized probes occurs via…
It depends on type of label used for the probe!
1) Autoradiography = If radiolabel was used
2) Photography (CCD) = If fluorophore was used
Northern Blot:
What information do we get from the band/signal detection step?
1) Whether a signal is PRESENT or ABSENT
(does the sample contain the RNA of interest?)
2) The STRENGTH of a signal == Reflects the AMOUNT of RNA present! == Reflect level of expression
(how much of the RNA of interest is present?)
In a Northern Blot after development, the presence of a band tells us that ________________ but the strength/intensity of that band tells us ___________
Presence of a band signal = RNA of interest is found in the sample
Strength of band signal = Relative amount of RNA of interest in the sample == Denotes level of gene expression!
