OCR Specification focus:
‘Explain base pairing and antiparallel strands forming the double helix; outline DNA precipitation.’
Deoxyribonucleic acid (DNA) is the molecule that stores genetic information in all living organisms. Its elegant double helix structure and reliable replication underpin inheritance, gene expression, and biological continuity across generations.
DNA Structure
The Composition of DNA
DNA is a polynucleotide, meaning it is a polymer made up of repeating nucleotide monomers. Each nucleotide in DNA is composed of three main components:
A deoxyribose sugar (a five-carbon pentose sugar lacking one oxygen atom compared to ribose).
A phosphate group attached to the sugar’s 5' carbon.
A nitrogenous base, which can be one of four: adenine (A), thymine (T), cytosine (C), or guanine (G).
The sugar and phosphate form the backbone of the DNA molecule, while the bases project inward and form pairs that connect the two strands.
Purines and Pyrimidines
The nitrogenous bases fall into two categories:
Purines: Adenine (A) and Guanine (G); these have a double-ring structure.
Pyrimidines: Thymine (T) and Cytosine (C); these have a single-ring structure.
The pairing between a purine and a pyrimidine maintains a consistent distance between the two DNA strands, preserving the helix’s uniform diameter.
Base Pairing Rules
Complementary base pairing is a defining feature of DNA structure. Hydrogen bonds form between specific pairs of bases, ensuring replication and transcription accuracy.
Complementary Base Pairing: The specific hydrogen bonding between adenine and thymine (A–T) through two hydrogen bonds, and between guanine and cytosine (G–C) through three hydrogen bonds.
These hydrogen bonds, while weak individually, collectively stabilize the DNA molecule. The higher the proportion of G–C pairs, the greater the DNA’s overall stability due to the extra bond.
The Double Helix
The two DNA strands twist around each other to form a double helix, first modelled by Watson and Crick in 1953 based on Rosalind Franklin’s X-ray diffraction data.
Each strand has a sugar-phosphate backbone running in opposite directions, creating an antiparallel configuration.
Antiparallel: The orientation of the two DNA strands, where one runs in a 5' to 3' direction and the complementary strand runs 3' to 5'.
This antiparallel nature is essential for the enzymatic activity during replication and transcription, as enzymes such as DNA polymerase can only synthesize new DNA in the 5' to 3' direction.
Two antiparallel polynucleotide strands run 5’→3’ and 3’→5’, held together by complementary base pairing to form the double helix.
A labelled diagram of the DNA double helix showing the sugar–phosphate backbones and complementary bases. The strands are antiparallel, running in opposite 5’→3’ directions. This visual reinforces the helical arrangement that results from base pairing and backbone geometry. Source.
Hydrogen Bonding and Helical Stability
The helical shape is further stabilized by:
Hydrogen bonds between base pairs.
Hydrophobic interactions between stacked base pairs.
The phosphate backbone’s negative charge, which allows interaction with positively charged proteins like histones.
The DNA molecule typically exists in the B-form, a right-handed helix with about 10 base pairs per turn.
Complementary base pairing means A pairs with T with two hydrogen bonds and G pairs with C with three hydrogen bonds.
A high-resolution NIH diagram of Watson–Crick base pairing. It shows two hydrogen bonds between A–T and three between G–C, with atoms and bonding indicated. This detail underpins helix stability and fidelity of genetic information. Source.
DNA Purification Practical
Purpose of the Practical
DNA purification, or DNA precipitation, is a core biological technique that allows students and scientists to extract visible DNA strands from biological material. This process isolates DNA by removing other cellular components such as proteins, lipids, and carbohydrates.
General Procedure for DNA Extraction and Precipitation
The DNA purification practical follows a series of key stages designed to release, separate, and precipitate DNA from cells.
1. Cell Lysis
The first step involves breaking open the cells to release their contents. This is achieved using a detergent solution.
Detergent: A chemical that breaks down cell and nuclear membranes by dissolving the lipid bilayer and disrupting protein–lipid interactions.
This exposes the DNA and other intracellular molecules.
2. Removal of Proteins and Enzymes
Once the cells are lysed, protease enzymes or salt solutions are added to degrade proteins and neutralize charges on the DNA molecule.
Protease digests histones and other associated proteins.
Sodium chloride helps the DNA strands aggregate by shielding the negative phosphate groups.
This stage ensures that the DNA remains intact and separate from other biomolecules.
3. Filtration
The mixture is filtered or centrifuged to remove cell debris and insoluble materials, leaving a clear solution containing dissolved DNA and other soluble components.
4. DNA Precipitation
The final stage involves adding ice-cold ethanol or isopropanol to the filtrate.
DNA Precipitation: The process of causing DNA to form a visible solid by reducing its solubility in an aqueous solution using alcohol.
DNA is insoluble in alcohol, especially at low temperatures. As the alcohol is poured carefully onto the surface of the solution, DNA precipitates at the interface and appears as white, stringy fibres.
In the practical, DNA is precipitated by carefully layering cold ethanol over the lysate so the DNA forms a white, stringy precipitate at the interface which can be spooled.
Key Points About the Process
Cold alcohol ensures minimal DNA degradation.
The DNA can be spooled using a glass rod for observation.
Care must be taken to avoid mixing the layers too vigorously, as this may shear the DNA strands.
Applications and Importance
DNA purification is essential for a wide range of modern biological studies and biotechnological applications, such as:
Genetic analysis, including DNA sequencing and PCR.
Forensic science, where DNA is extracted from minute biological samples.
Molecular cloning, where purified DNA is inserted into vectors for genetic engineering.
A clear understanding of this practical reinforces theoretical knowledge of DNA’s chemical structure and properties.
Connection to the Specification
This sub subtopic links structural theory with practical laboratory skills. Students must be able to explain base pairing, understand antiparallel strands forming the double helix, and outline the process of DNA precipitation as an applied example of molecular biology.
FAQ
The uniform width of DNA results from base pairing between one purine and one pyrimidine. Purines (A and G) are larger, double-ringed molecules, while pyrimidines (T and C) are smaller, single-ringed ones.
When a purine always pairs with a pyrimidine, the total distance across the two strands remains constant. This consistent geometry is essential for the regular twisting of the double helix, allowing enzymes like DNA polymerase to interact with DNA efficiently during replication.
Several factors influence how much DNA precipitates and how visible it is:
Temperature: Ice-cold ethanol maximises precipitation by reducing DNA solubility.
Ethanol concentration: Too dilute or warm ethanol reduces yield.
Mixing speed: Vigorous mixing can shear long DNA strands, making them less visible.
Salt concentration: Insufficient salt leaves negative phosphate groups unshielded, reducing aggregation.
Precision in these conditions ensures clean, intact DNA suitable for observation.
DNA is highly soluble in water because of its charged phosphate groups, which interact readily with polar water molecules.
Ethanol is less polar and disrupts these interactions. In the presence of salt, DNA becomes insoluble and clumps together, forming visible fibres. The use of ice-cold ethanol slows down molecular motion, reducing degradation and increasing yield.
Warm or impure ethanol would keep DNA partially dissolved, resulting in a faint or no precipitate.
The antiparallel orientation (5’→3’ and 3’→5’) is crucial for enzyme function and replication accuracy.
DNA polymerase can only synthesise new strands in the 5’→3’ direction. The antiparallel setup allows one strand (the leading strand) to be synthesised continuously and the other (the lagging strand) discontinuously.
It also ensures that the hydrogen bonds between complementary bases align correctly, maintaining helix stability.
Without this arrangement, enzymes would not attach or read the template strand properly
Though simple, DNA purification involves materials requiring safe handling and ethical awareness:
Detergents and enzymes can irritate skin or eyes — gloves and goggles must be worn.
Ethanol is flammable and should be kept away from flames or heat sources.
When using biological materials (e.g. onion tissue or cheek cells), ethical care ensures that samples are obtained responsibly and disposed of hygienically.
Good laboratory practice and risk assessment are essential for valid, safe experimental outcomes.
Practice Questions
Question 1 (2 marks)
Describe how base pairing contributes to the structure of the DNA double helix.
Mark scheme:
1 mark: States that adenine pairs with thymine and guanine pairs with cytosine.
1 mark: Explains that hydrogen bonds between complementary bases hold the two antiparallel strands together in the double helix.
Question 2 (5 marks)
Describe the main steps involved in the DNA purification (precipitation) practical and explain the purpose of each step.
Mark scheme:
1 mark: Cell lysis — detergent breaks down cell and nuclear membranes to release DNA.
1 mark: Protease or salt addition — removes proteins or neutralises DNA’s negative charges to allow strands to aggregate.
1 mark: Filtration — separates cell debris from soluble DNA.
1 mark: Addition of cold ethanol (or isopropanol) — reduces DNA solubility so it precipitates as visible strands.
1 mark: Observation/spooling — DNA appears as white, stringy fibres that can be collected using a glass rod.
