V 1 1 June 2018 Copyright by Amplyus

Science for everyone, everywhere Patent pending We bring the study of DNA structure to your hands DNA GLOW Lab

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

The DNA model Science for everyone, everywhere “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 Ph. D 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. • Photo 51, an X-Ray diffraction image made by Raymond Gosling under the direction of Rosalind Franklin, suggested that DNA was a double helix. DNA GLOW Lab

The DNA model Science for everyone, everywhere • 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. DNA GLOW Lab

Purines and pyrimidines Science for everyone, everywhere 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. DNA GLOW Lab

Base pairing Science for everyone, everywhere A single ring pyrimidine will always bind to a double ring purine giving DNA its constant width. Hydrogen bonds hold the two strands together. DNA GLOW Lab

Base pairing Science for everyone, everywhere Hydrogen bonds are weaker than covalent bonds and are more subject to changes in physical and chemical properties such as heat and p. H. DNA GLOW Lab

Base pairing Science for everyone, everywhere When hydrogen bonds are heated, they will break, and DNA will denature. When cooled, they will reform, and DNA will anneal. DNA GLOW Lab

Science for everyone, everywhere 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. DNA GLOW Lab

Equipment needed Science for everyone, everywhere Heat source Or Blue light illuminator & DNA GLOW Lab Or

Determining GC content Science for everyone, everywhere Can you determine which sample has the highest GC content? The lowest? DNA GLOW Lab

Determining GC content Science for everyone, everywhere Heat to 95˚. Wait 1 minute. Quickly transfer to blue light illuminator. *Continue viewing for about one minute. DNA GLOW Lab

Programming the mini. PCR Science for everyone, everywhere Choose Library Choose +NEW Choose Create Heat Block Protocol DNA GLOW Lab

Programming the mini. PCR Science for everyone, everywhere Give it an awesome and creative name Choose 95˚ Save and run DNA GLOW Lab

Determining GC content Science for everyone, everywhere Heat to 95˚. Wait 1 minute. Quickly transfer to blue light illuminator. *Continue viewing for about one minute. DNA GLOW Lab

Determining GC content Science for everyone, everywhere Which sample had the highest GC content? The lowest? Justify your answer. DNA GLOW Lab

GC Rich DNA has more bonds Science for everyone, everywhere AT Rich Hydrogen bonds work like little magnets. The more bonds, the more attraction between the two strands GC Rich DNA GLOW Lab

Melting temperature Science for everyone, everywhere GOAL: • Identify approximate melting temperatures for each tube 1. Proram the mini. PCR for a linear ramp from 35°-99°. This will cause the machine to slowly heat over time. 2. Place tubes in wells. *Be careful. Lid and heat block will be hot. 3. Every two minutes, transfer tubes to illuminator. 4. Take picture and return to heat block. DNA GLOW Lab

Programming the mini. PCR Science for everyone, everywhere Choose +NEW Choose Create Linear Ramp Protocol DNA GLOW Lab

Programming the mini. PCR Science for everyone, everywhere Give it an awesome and creative name Start @ 35˚ End @ 99˚ Time 10 min Save and run DNA GLOW Lab

Melting temperature Science for everyone, everywhere Results? DNA GLOW Lab

p. H and hydrogen bonds Science for everyone, everywhere OH Alkaline (high p. H) conditions have an excess of OH- ions in solution. OH The excess negative charges will be attracted to the positively charged hydrogen atoms in the hydrogen bonds. OH With the hydrogen atoms now bonding with the OH- ions in solution, the DNA will denature. OH OH DNA GLOW Lab

p. H and hydrogen bonds Science for everyone, everywhere Create a p. H dilution series using Na. OH to test the effect of alkaline conditions on hydrogen bonding between nucleotides. Is this effect reversible? How could you test that? DNA GLOW Lab

DNA concentration Science for everyone, everywhere 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. DNA GLOW Lab