Nucleic Acids (Detection and Quantification) Flashcards
Want information can we get from Qualitative analysis of nucleic acids?
- The nature of the molecules
- Size
- Nucleotide composition (sequence)
- Conformation/Configuration
- Structure
What information can we get from Quantitative analysis of nucleic acids?
Levels of gene products / The abundancy
(In specific environments, for understanding cancer)
What is a probe?
It is a specific base-pairing sequence complementary to the sequence of interest
What is another name for a short probe? (short sequence of amino acids)
oligonucleotide
What is the role of Polynucleotide Kinase (PNK)?
Labelling the oligonucleotide/probes to be able to identify them (by phosphorylating nucleotides)
Gamma phosphate on ATP transfers onto the 5’ end of the oligonucleotide
What method can be used to make a labelled DNA prob? (Double stranded)
Explain.
PCR:
1. Incorporate sNTPs that carry isotopic radiolabel on alpha-phosphate (bc beta and gamma leave)
ex: dGTP, dTTP, dATP, dCTP (in a lower concentration), (a-32P dCTP)
2. Run PCR
3. Wash off extra radioactive dNTPs
4. Before use, denature the double strands bc single strands needed to bind to target regions !!!!
What are the steps for analysis of DNA by transfer on a solid state support?
- Cleave DNA into segments with restriction enzyme
- Blotting technique to separate segments according to size (in agarose gel)
- Alkaline solution to denature DNA (want single stranded)
- Capillary action transfers DNA from agarose gel onto blot/solid state support (nylon or nitrocellulose)
- Dry it out
- Get permanent record of segments (single stranded)
- Use Probe (if DS probe, denature 1st by boiling)
- Wash off unbound probes
- See where the prob is located on the gel separated solid support by radioactive detector
What is the main difference between the steps in DNA and RNA analysis by transferring to solid state support?
RNA coils into weird conformations so need to be denatured before is it past in the agarose gel to not migrate according to structure
Do everything in denaturing conditions
What are the possible solid state support?
How can nucleic acids that are already strongly bound to it can be so permanently?
Possible supports = Nylon, Nitrocellulose
Nucleic acids bind permanently by UV crosslinking to have permanent record
What does it mean for a blot to be hyrbidized with something?
Blot = solid support
To be hybridized with probes means that it is exposed to probes that bind to it → Can see where probes bonded on Autoradiogram
How can polymorphisms be detected without probes?
- Restriction enzymes are used to cut specific recognized sequences to have multiple segments
- If after exposure to restriction enzyme, we still have only 1 size on the blot, the gene wasn’t cut, which means there is a mutation in the restriction recignition site.
What are the differences between a Southern blot, a Western blot and a Northern blot?
Southern blot = analysis of DNA
Western blot = analysis of proteins
Northern blot = analysis of RNA
What can southern blot be used for?
To identify genes related with diseases and its presence in different members of a family
What characteristics of RNA can be analyzed with a Northern Analysis?
Northern analysis = gel electrophoresis + RNA segments
- Tissue-specific expression
- Stage-specific/ temporal expression (if run 2 of them at different stages of dev.)
(If you know how many pmols associated with your probe, can get estimate of how many copies of RNA on each level with intensity of radioactivity
For a probe to identify its ligand does it have to be 100% complementary all the way?
Nope, just need enough length and enough complementary regions
What is quantitative RT-PCR? and what does it allows us to determine (2 words)?
RT-qPCR = reverse transcriptase quantitative polymerase chain reaction
Allows to determine mRNA levels
What is the process involved in RT-qPCR?
- Reverse transcriptase makes SS-DNA complementary from mRNA sample
Primer specific mRNA of interest with specific primer - Synthesis of the second strand to get cDNA
- quantitative PCR = PCR mix (Taq + DNAP + dNTP) + fluorescent dye that only fluorescences when it collides to DS-DNA
What is the main difference between normal reverse transcription and reverse transcription in the context of RT-qPCR?
Normal RT: Use of a Poly dT primer (complementary to mRNA poly A tail present on ALL mRNAs)
RT-qPCR: Primer = sequence that recognizes one specific mRNA of the sample given
What are the phases of PCR reactions?
What influences the rate of these phases?
- Ground phase
- Exponential phase
- Linear phase
- Plateau phase: reached in much fewer cycle in samples with greater amouts of starting material (cDNA)
The amount of cDNA in a sample directly proportional to abundance of mRNA in original sample
What is the Quantitative Cycle of a qPCR reaction?
It is the number of the cycle at which the reaction enters the exponential phase
Explain the whole process of Reverse Transcriptase?
- Pimer = poly T oigonucleotide complementary to the 3’ poly A tail
- Synthesis of the 1st DNA strand complementary to mRNA template
- RNA template removed and poly G adapter annealed to the 3’ end
- Poly dC primer used to initiate synthesis of 2nd DNA strand
- E. coli DNA pol I synthesizes the 2nd DNA strand
What is the benefit of a cDNA library?
It is a stable permanent record of all mRNA present in the tissue (Can keep permanently by putting in vector)
What is the process of RNA-seq?
- Take a cell and extract all its RNA
- Isolate mRNA by size selection (gel) or Poly-A selection (only mRNAs have poly A tails)
- By Reverse transcriptase, convert mRNA → cDNA
- Ligation of adaptors for Next-Gen Seq
- PCR amplification
- Sequencing (with genome alignment and quantification)
What are the main differences between RT-qPCR and RNA-seq?
RNA-seq provides view of entire transcriptome (all mRNAs in sample)
RT-sPCR measures expression of specific target gene
What are the major regulators of gene expression?
- Rate of transcription (MAJOR ONE)
- mRNA translation
- Protein degradation
- mRNA degradation
By what is symbolized the transcription start site?
at +1 site with an arrow