1. A new way to investigate DNA structure.
V1.1: June 2018
© Copyright by Amplyus LLC, all rights reserved
DNA GLOW Lab
A new way to investigate DNA structure.

2. We bring the study of DNA structure to your hands
Patent pending
We bring the study of DNA structure to your hands
Sell harder first.

3. Goals for today: DNA GLOW Lab
Review DNA structure and chemical properties.
Predict how DNA will respond to changes in its chemical and physical environment.
Test predictions using a fluorescent dye that allows us to visually verify aspects of DNA structure.

4. The DNA model
“This structure has novel features which are of considerable biological interest.”
- A Structure for Deoxyribose Nucleic Acid Watson J. and Crick F Nature 171, 737 (1953)
In 1952, 23-year-old James Watson and 37-year-old PhD student Francis Crick built the first accurate model of DNA.
They based their model on several sources. Notably:
Data from Erwin Chargaff suggested that nucleotides were bound in pairs, adenine to thymine and guanine to cytosine.
Photo51, an X-Ray diffraction image made by Raymond Gosling under the direction of Rosalind Franklin, suggested that DNA was a double helix.

5. The DNA model
Today, the double helix is an iconic image in science that we are all familiar with.
Like all molecules, however, its structure had to be deduced using chemical tests.
We now know that DNA is made of two backbones comprised of alternating five-carbon sugars and phosphate groups wrapped around nitrogenous bases which are joined by hydrogen bonds.
This proposed structure leads to predictions about the molecule’s chemical properties that we can test in the laboratory.
-This is the structure of the double helix- two strands joined by hydrogen bonds.
-Using fluorescent dyes we will be able to visualize changes made to that structure in the classroom.
-Investigate the conditions that will lead to changes in DNA structure.
-Dye binds to double stranded DNA, but not single Stranded.

6. Purines and pyrimidines
Purines (adenine and guanine) have a double ring structure.
Pyrimidines (thymine and cytosine) have a single ring structure.
Guanine and cytosine can make three bonds.
Adenine and thymine can make two bonds.
The polarity of the bonds between the homologous pairs is reversed.
-Quick refresher on base pairing.
-Purines A and G double ring.
-Pyrimidines C and T single rings.
-GC pairs make three H bonds
-AT pairs make 2 H bonds

7. Hydrogen bonds hold the two strands together.
Base pairing
-Complimentary base pairing the basis for understanding DNA Structure.
-Purines with pyrimidines maintains constant width of the double helix.
-Number of H bonds determines AT and GC
A single ring pyrimidine will always bind to a double ring purine giving DNA its constant width.
Hydrogen bonds hold the two strands together.

8. Base pairing
Hydrogen bonds are weaker than covalent bonds and are more subject to changes in physical and chemical properties such as heat and pH.
Bonds are relatively weak and can be broken by changing physiochemical conditions.
Temperature. Raising the temperature will cause H bonds to break and DNA will become single stranded.

9. Base pairing
When hydrogen bonds are heated, they will break, and DNA will denature. When cooled, they will reform, and DNA will anneal.
-Cooling DNA will do the opposite. At a specific temperature H bonds will reform and DNA will become double stranded again.
-Basis of many biotech applications – especially PCR.

10. Exploring DNA Structure
DNA GLOW Lab:
Exploring DNA Structure
DNA is mixed with a dye that fluoresces only when bound to double stranded DNA.
When physical or chemical conditions are sufficiently changed, DNA will become denatured and will no longer fluoresce.
When conditions are returned, DNA will anneal and fluorescence will return.

11. Blue light illuminator
Equipment needed
Heat source
Blue light illuminator
& Or Or
- Temp (water bath or thermocycler)
Thermocylcer. Need to heat tubes quickly to precise temperatures.
-Water bath.

12. Can you determine which sample has the highest GC content?
Determining GC content
Can you determine which sample has the highest GC content?
The lowest?

13. Determining GC content
Heat to 95˚.
Wait 1 minute.
Quickly transfer to blue light illuminator.
-Goal 1: Which is the AT, CG and 50/50 tubes.
Hint that there wiklk be a difference
*Continue viewing for about one minute.

14. Choose Create Heat Block Protocol
Programming the miniPCR
Choose Library
Choose +NEW
Choose Create Heat Block Protocol

15. Give it an awesome and creative name
Programming the miniPCR
Give it an awesome and creative name
Choose 95˚
Save and run

16. Determining GC content
Heat to 95˚.
Wait 1 minute.
Quickly transfer to blue light illuminator.
-Goal 1: Which is the AT, CG and 50/50 tubes.
Hint that there wiklk be a difference
*Continue viewing for about one minute.

17. Which sample had the highest GC content?
Determining GC content
Which sample had the highest GC content?
The lowest?
Justify your answer.

18. AT Rich GC Rich GC Rich DNA has more bonds
Hydrogen bonds work like little magnets. The more bonds, the more attraction between the two strands
-You’ll notice there were three tubes.
-Replace GC on the top strand with AT
-Replace AT on the bottom strand with GC
-What is different between strands? # of bonds.
-How will that affect the temperature?
GC Rich

19. Melting temperature GOAL:
Identify approximate melting temperatures for each tube
Proram the miniPCR for a linear ramp from 35°-99°. This will cause the machine to slowly heat over time.
Place tubes in wells *Be careful. Lid and heat block will be hot.
Every two minutes, transfer tubes to illuminator.
Take picture and return to heat block.
-Depending on time teacher can play for a couple minutes.
-Goal 2: Find approximate melting temp through trial and error.
-Try to find temperatures that will denature some, but not all of the tubes.
-Allow to play , but need to be done by about 37 minutes into time block.

20. Choose Create Linear Ramp Protocol
Programming the miniPCR
Choose +NEW
Choose Create Linear Ramp Protocol

21. Give it an awesome and creative name
Programming the miniPCR
Give it an awesome and creative name
35˚
99˚
Time 10 min
Save and run

22. Melting temperature
Results?
Data sheet?

23. pH and hydrogen bonds OH OH OH OH OH
Alkaline (high pH) conditions have an excess of OH- ions in solution.
The excess negative charges will be attracted to the positively charged hydrogen atoms in the hydrogen bonds.
With the hydrogen atoms now bonding with the OH- ions in solution, the DNA will denature. OH OH OH OH

24. Is this effect reversible? How could you test that?
pH and hydrogen bonds
Create a pH dilution series using NaOH to test the effect of alkaline conditions on hydrogen bonding between nucleotides.
Is this effect reversible? How could you test that?

25. DNA concentration
Fluorescent dyes are often used in the laboratory to measure DNA concentration.
Because the dye only fluoresces when bound to double stranded DNA, an accurate concentration can be measured even in a complex mixture containing other molecules.
In the laboratory, normally a tool called a fluorometer would give an accurate reading of fluorescence intensity.

26. DNA concentration
Create a DNA dilution series to measure the concentration of the unknown DNA sample.

27. Thank You!
Feedback?
Questions?
@miniPCR