Nucleic Acid Labelling Flashcards
Draw a segment of DNA showing the 5’ phosphate end and the 3’ OH end and internal phosphates
List THREE places where DNA can be labelled
- 5’ end
- 3’ end
- Internally
Explain how a radioactive phosphate can be added using an enzymatic approach to the 5’ end of a chemically synthesised oligonucleotide
- Synthesised with no 5’ phosphate
- Kinase + dATP with labelled gamma phosphate added to 5’ end
Explain how a radioactive phosphate can be added using an enzymatic approach to the 5’ end of a a native strand of DNA
- 5’ phosphate removed by phosphatase
- Kinase + dATP with labelled gamma phosphate added to 5’ end
Explain how a radioactive phosphate can be incorporated into DNA
- Primer attached to DNA strand
- DNA Pol + dATP with labelled alpha phosphate + dCTP + dGTP + dTTP added to end of primer
Explain how a fluorescent molecule can be added to a DNA molecule at the 5’ or 3’end
- A Fluorescent Base
OR - A Fluorophore via a Linkage
- Phosphoramidite Method
Explain how a fluorescent molecule can be incorportated into a DNA molecule
By attaching fluorescent dye to oligonucleotide sequences at base positions that don’t interfere with hydrogen bonding
What is the absorption and emission spectra of 6-FAM and TAMRA?
- 6-FAM = 494nm
- TAMRA = 565nm
What is a DNA chain elongation inhibitor?
- Terminate chain elongation in DNA sequencing
- Common terminators are ddATP, ddCTP, ddGTP, ddTTP
How does ddTTP cause chain termination of DNA synthesis?
ddNTP do not have a free 3’ OH group
Explain how fluorescent chain elongation inhibitors can be used to sequence DNA
- Stops DNA elongation
- DNA made into single strand and scanned for fluorescence
- Scan shows that DNA extension terminated with a ddNTP
- Each of the four ddNTPs are a different colour so can be distinguished from each other and DNA sequence can be read
- Sanger Sequencing
Why can’t a newly synthesised double stranded oligonucleotide be ligated to another newly synthesised double stranded oligonucleotide?
- Usually a 5’ phosphate is added after chemical synthesis
- Absence of 5’ phosphate prevents ligation of the strands
How would you attach an oligonucleotide to a solid surface?
Using a Thiol C6 S-S
Why might you make an oligonucleotide with an amino modifier?
To attach oligos to solid surfaces or to another molecule
Why might you make an oligonulceotide with a thiol C6 S-S?
To link oligos to solid surfaces
Describe the avidin-biotin interaction
(write a mini essay)
How many biotin can avidin bind?
4
How can biotin be added to the 5’ or 3’ end of an oligonucleotide?
Synthesis of Biotinylated Oligonucleotides
How can biotin be incorportated into DNA?
By using biotin dT or amino bases for conjugation to biotin-NHS
What is the Kd for avidin-biotin binding?
10^-15
What are the features of avidin-biotin binding that make it so useful?
- Rapid bond formation
- Bond unaffected by extreme pH, temperature, organic solvents, and other denaturing agents
List FIVE applications for which the avidin-biotin interaction is used
- ELISA
- Immunohistochemistry (IHC)
- Western Blotting
- Cell Surface Labelling
- Immunoprecipitation
How are antibodies raised against an antigen?
- Animal innoculated with specific antigen
- Animals immune response causes B-cells to produce antibodies against antigen
- Antibodies purified from plasma
How are antibodies bound directly and indirectly to surfaces?
Describe an antibody-biotin-streptavidin-enzyme based chemiluminescent detection assay
Use diagrams to illustrate answer
- Antibody biotinylated with streptavidin
- Streptavidin linked to enzyme
- Antigen stuck to surface of microplate well
- Antibody sticks to antigen
- 3 biotinylated enzymes bound to antibody via streptavidin
- Enzymes convert substrate to fluor product
- Detection of fluorescence associated with levels of antigen present
Explain how avidin-biotin interaction can be used to capture a protein linked to a promoter
- Primer designed to bind gene of interest on DNA
- Biotin molecule attached to oligo-NT primer
- Beads attached to the biotin molecule after elongation
- Bead increases MW of oligo
- Centrifugation used to form bead-oligo complex pellet
OR - Bead made from Fe+ and a magnet used to isolate DNA
- Protein removed using salt precipitation
- Protein identified using SDS-PAGE and Western Blotting
Explain the principle of the electrophoretic mobility shift assay (EMSA)
AIM:
* Used to determine if a protein binds to DNA
* Protein bound to DNA will retard its migration in gel electrophoresis
METHOD:
* DNA fragment is synthesised
* DNA is labelled by adding a fluorescent or radioactive molecule to 5’ end
* Labelled DNA incubated
* Sample loaded on a polyactrylamide gel and electrophoresis carried out
* Gel scanned to detect labelled DNA
* Compared to migration distances of control sample
* If test sample binds to DNA, its migration through gel will be retarded compared to control
Explain the principle of DNA footprinting
AIM:
* To determine the exact sequences in a DNA fragment to which a protein binds
* Protein bound to DNA protects it from enzymes that cleave or modify DNA
METHOD:
* ssDNA fragment labelled at 5’ end with fluorescent or radioactive label
* Annealed into dsDNA
* Labelled dsDNA fragment incubated with protein sample
* Identical sample with no protein used as a control
* Cleavage agent that cleaves the phosphodiester bond is added at limiting concentrations to that 1 nick is introduced per single DNA molecule
* DNA denatured into single strands and separated based on size by polyacrylamide gel electrophoresis
* Labelled DNA detected by scanning gel for fluorescence
* Site of protein binding revealed by comparing pattern of fragments in control to test sample
* DNA sequence ladder run alongside sample in electrophoresis to allow matching of DNA bases protected from cleavage with exact sequence of DNA
Explain the principle of the Chromatin Immunoprecipitation (ChIP) assay
AIM:
* Study protein-DNA interaction in vivo
* Proteins / transcription factors bind to DNA in the nucleus in a chromatin context
METHOD:
* Agents such as formaldehyde or UV light used to cross-link proteins to the DNA
* After cross-linking, DNA with cross-linked protein is purified and fragmented
* Antibody that recognises protein of interest is added to mixture
* Antibody binds to protein of interest which has DNA bound to it
* Second antibody linked to beads that recognises first antibody is added to mixture
* Beads are isolated by centrifugation and cross-link is dissolved
* DNA bound to captured protein is released
* Released DNA amplified with PCR using specific primers
* Once amplified, DNA is sequenced to identify exact sequence bound by protein
* ChIP method adapted to investigate all protein binding sites in genome
What is a spacer molecule used for?
Increases distance between oligonucleotide and a conjugated modification
What is Inosine?
A nucleoside that can pair with all other bases (A, T, G, C)
Outline a use for Inosine
When complementary BP is unknown in a DNA amplification
Why might cholesterol be added to an oligonucleotide?
To allow passage of oligonucleotide through plasma membrane
How could you make an oligonucleotide resistant to nuclease degradation?
Use 2’-OMe-nucleoside-containing oligonucleotides
How could you stop mRNA from provoking an immune response?
Modify Uridine to Pseudouridine
What is peptide nucleic acid?
A synthetic analogue of DNA in which the ribose phosphate backbone has been replaced by a polyamide chain
Outline the use for peptide nucleic acid
- Binds complementary DNA/RNA with higher affinity and greater specificity
- PNA is more stable as they arae resistant to nucleases
Explain how nick translation works - use diagrams to illustrate answer
Tagging technique where DNA polymerase I is used to replace some of the nucleotides of a DNA sequence with labelled analogues
- DNA molecules treated with DNAse to produce single-stranded “nicks”
- DNA Pol I elongates 3’ hydroxyl terminus of nicked sites, removing nucleotides by 5’-3’ exonuclease activity
- Removed nucleotides replaced with modified dNTPs, thus labelling DNA molecule
What is the difference between using random primers vs. oligo dT primers vs. region specific primers in making cDNA from mRNA using reverse transcriptase?
- Random Primers - bind throughout entire RNA template
- Oligo dT Primers - bind to template so that entire template RNA is transcibed into cDNA
- Region Specific Primers - Bind to an intended site so that desired transcript can be converted to cDNA
