Nucleic Acid Labelling

Question 1

Draw a segment of DNA showing the 5’ phosphate end and the 3’ OH end and internal phosphates

Answer 1

Question 2

List THREE places where DNA can be labelled

Answer 2

1. 5’ end
2. 3’ end
3. Internally

Question 3

Explain how a radioactive phosphate can be added using an enzymatic approach to the 5’ end of a chemically synthesised oligonucleotide

Answer 3

• Synthesised with no 5’ phosphate
• Kinase + dATP with labelled gamma phosphate added to 5’ end

Question 4

Explain how a radioactive phosphate can be added using an enzymatic approach to the 5’ end of a a native strand of DNA

Answer 4

• 5’ phosphate removed by phosphatase
• Kinase + dATP with labelled gamma phosphate added to 5’ end

Question 5

Explain how a radioactive phosphate can be incorporated into DNA

Answer 5

• Primer attached to DNA strand
• DNA Pol + dATP with labelled alpha phosphate + dCTP + dGTP + dTTP added to end of primer

Question 6

Explain how a fluorescent molecule can be added to a DNA molecule at the 5’ or 3’end

Answer 6

• A Fluorescent Base
  OR
• A Fluorophore via a Linkage
• Phosphoramidite Method

Question 7

Explain how a fluorescent molecule can be incorportated into a DNA molecule

Answer 7

By attaching fluorescent dye to oligonucleotide sequences at base positions that don’t interfere with hydrogen bonding

Question 8

What is the absorption and emission spectra of 6-FAM and TAMRA?

Answer 8

• 6-FAM = 494nm
• TAMRA = 565nm

Question 9

What is a DNA chain elongation inhibitor?

Answer 9

• Terminate chain elongation in DNA sequencing
• Common terminators are ddATP, ddCTP, ddGTP, ddTTP

Question 10

How does ddTTP cause chain termination of DNA synthesis?

Answer 10

ddNTP do not have a free 3’ OH group

Question 11

Explain how fluorescent chain elongation inhibitors can be used to sequence DNA

Answer 11

• 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

Question 12

Why can’t a newly synthesised double stranded oligonucleotide be ligated to another newly synthesised double stranded oligonucleotide?

Answer 12

• Usually a 5’ phosphate is added after chemical synthesis
• Absence of 5’ phosphate prevents ligation of the strands

Question 13

How would you attach an oligonucleotide to a solid surface?

Answer 13

Using a Thiol C6 S-S

Question 14

Why might you make an oligonucleotide with an amino modifier?

Answer 14

To attach oligos to solid surfaces or to another molecule

Question 15

Why might you make an oligonulceotide with a thiol C6 S-S?

Answer 15

To link oligos to solid surfaces

Question 16

Describe the avidin-biotin interaction
(write a mini essay)

Question 17

How many biotin can avidin bind?

Answer 17

4

Question 18

How can biotin be added to the 5’ or 3’ end of an oligonucleotide?

Answer 18

Synthesis of Biotinylated Oligonucleotides

Question 19

How can biotin be incorportated into DNA?

Answer 19

By using biotin dT or amino bases for conjugation to biotin-NHS

Question 20

What is the Kd for avidin-biotin binding?

Answer 20

10^-15

Question 21

What are the features of avidin-biotin binding that make it so useful?

Answer 21

• Rapid bond formation
• Bond unaffected by extreme pH, temperature, organic solvents, and other denaturing agents

Question 22

List FIVE applications for which the avidin-biotin interaction is used

Answer 22

• ELISA
• Immunohistochemistry (IHC)
• Western Blotting
• Cell Surface Labelling
• Immunoprecipitation

Question 23

How are antibodies raised against an antigen?

Answer 23

• Animal innoculated with specific antigen
• Animals immune response causes B-cells to produce antibodies against antigen
• Antibodies purified from plasma

Question 24

How are antibodies bound directly and indirectly to surfaces?

Answer 24

Question 25

Describe an antibody-biotin-streptavidin-enzyme based chemiluminescent detection assay Use diagrams to illustrate answer

Answer 25

* 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

Question 26

Explain how avidin-biotin interaction can be used to capture a protein linked to a promoter

Answer 26

* 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

Question 27

Explain the principle of the electrophoretic mobility shift assay (EMSA)

Answer 27

**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

Question 28

Explain the principle of DNA footprinting

Answer 28

**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

Question 29

Explain the principle of the Chromatin Immunoprecipitation (ChIP) assay

Answer 29

**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

Question 30

What is a spacer molecule used for?

Answer 30

Increases distance between oligonucleotide and a conjugated modification

Question 31

What is Inosine?

Answer 31

A nucleoside that can pair with all other bases (A, T, G, C)

Question 32

Outline a use for Inosine

Answer 32

When complementary BP is unknown in a DNA amplification

Question 33

Why might cholesterol be added to an oligonucleotide?

Answer 33

To allow passage of oligonucleotide through plasma membrane

Question 34

How could you make an oligonucleotide resistant to nuclease degradation?

Answer 34

Use 2'-OMe-nucleoside-containing oligonucleotides

Question 35

How could you stop mRNA from provoking an immune response?

Answer 35

Modify Uridine to Pseudouridine

Question 36

What is peptide nucleic acid?

Answer 36

A synthetic analogue of DNA in which the ribose phosphate backbone has been replaced by a polyamide chain

Question 37

Outline the use for peptide nucleic acid

Answer 37

* Binds complementary DNA/RNA with **higher affinity** and **greater specificity** * PNA is **more stable** as they arae **resistant** to **nucleases**

Question 38

Explain how nick translation works - use diagrams to illustrate answer

Answer 38

Tagging technique where DNA polymerase I is used to replace some of the nucleotides of a DNA sequence with labelled analogues 1. DNA molecules treated with DNAse to produce single-stranded "nicks" 2. DNA Pol I elongates 3' hydroxyl terminus of nicked sites, removing nucleotides by 5'-3' exonuclease activity 3. Removed nucleotides replaced with modified dNTPs, thus labelling DNA molecule

Question 39

What is the difference between using random primers vs. oligo dT primers vs. region specific primers in making cDNA from mRNA using reverse transcriptase?

Answer 39

* **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