Difficulty: Intermediate | Prerequisites: Central dogma basics (DNA, RNA, protein)
This set of notes covers the physical structure of DNA, how cells replicate and repair it, and the laboratory tools scientists use to cut, paste, and edit DNA. These concepts span Module II of BIO 311C and form the foundation for understanding both molecular biology and modern biotechnology. You should be comfortable with the idea that DNA carries genetic information before starting here.
DNA is a double-stranded, antiparallel helix held together by complementary base pairing (A with T, G with C). Cells copy it with high fidelity using DNA polymerase, which also proofreads for errors, and repair pathways fix damage that slips through. Scientists exploit this chemistry with tools like restriction enzymes, plasmids, and CRISPR-Cas9 to cut, clone, and edit genes in the laboratory.
Hershey-Chase experiment
The 1952 experiment that confirmed DNA, not protein, is the molecule of heredity. Hershey and Chase used radioactive phosphorus (found in DNA) and radioactive sulphur (found in protein) to track which molecule entered bacterial cells during viral infection. Only phosphorus was found inside the cells.
Phosphodiester bond
The covalent bond that links one nucleotide to the next in a DNA or RNA strand, connecting the 3' carbon of one sugar to the 5' carbon of the next via a phosphate group. Think of it as the backbone of the strand.
Antiparallel
The orientation of the two strands of DNA: one runs 5' to 3', and the complementary strand runs 3' to 5'. In simple terms, the two strands point in opposite directions, like two lanes of traffic on a road.
Purine
A double-ringed nitrogenous base. In DNA, the purines are adenine (A) and guanine (G). The mnemonic "pure as gold" links purines to A and G.
Pyrimidine
A single-ringed nitrogenous base. In DNA, the pyrimidines are cytosine (C) and thymine (T). A purine always pairs with a pyrimidine, keeping the helix width constant.
Deoxyribose
The five-carbon sugar in DNA nucleotides. It differs from ribose (found in RNA) by lacking an oxygen atom on the 2' carbon. Think of it as the "D" in DNA.
DNA proofreading
The ability of DNA polymerase to detect and correct mismatched bases during replication. The enzyme reverses direction, removes the incorrect nucleotide, and inserts the correct one.
DNA repair pathway
Cellular mechanisms that detect and fix DNA damage after replication is complete. These include mismatch repair, excision repair, and double-strand break repair. They are the cell's quality-control system for its genome.
Restriction enzyme
A bacterial enzyme that cuts DNA at specific short recognition sequences. The cuts often produce "sticky ends," short single-stranded overhangs that can pair with complementary overhangs from another DNA fragment.
Sticky ends
The short, single-stranded DNA overhangs left after a restriction enzyme cuts. They are called "sticky" because their exposed bases can hydrogen-bond with complementary sticky ends, allowing researchers to join DNA fragments from different sources.
Plasmid
A small, circular piece of DNA found in bacteria, separate from the chromosomal DNA. Plasmids replicate independently and are widely used as vectors in genetic engineering to carry genes of interest into host cells.
Selectable marker
A gene inserted into a plasmid that allows researchers to identify which cells have taken up the plasmid. Common examples include antibiotic resistance genes and fluorescence genes. Think of it as a tag that says "this cell got the plasmid."
Origin of replication
The specific DNA sequence on a plasmid (or chromosome) where replication begins. A plasmid must have one to replicate inside a host cell.
Promoter
The DNA sequence upstream of a gene where RNA polymerase binds to begin transcription. In a plasmid, the promoter drives expression of the inserted gene of interest.
CRISPR-Cas9
A genome-editing system derived from bacterial immune defences. It uses a guide RNA to direct the Cas9 enzyme to a specific DNA sequence, where Cas9 cuts both strands. In simple terms, it is a programmable molecular scissors.
DNA consists of two polynucleotide strands wound around each other in a double helix.
Each nucleotide contains a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine (A), thymine (T), guanine (G), or cytosine (C).
The sugar-phosphate backbone runs along the outside; the bases face inward and form hydrogen bonds with their complementary partner.
Base pairing rules: A pairs with T (two hydrogen bonds), G pairs with C (three hydrogen bonds). A purine always pairs with a pyrimidine.
The two strands are antiparallel: one runs 5' to 3', the other 3' to 5'.
Phosphodiester bonds connect adjacent nucleotides within each strand.
Alfred Hershey and Martha Chase (1952) used bacteriophages (viruses that infect bacteria) to determine whether DNA or protein carries genetic information.
They labelled one batch of phages with radioactive phosphorus-32 (which marks DNA, since protein contains no phosphorus) and another with radioactive sulphur-35 (which marks protein, since DNA contains no sulphur).
After infection, only phosphorus-32 was found inside the bacterial cells. Sulphur-35 remained outside.
Conclusion: DNA, not protein, is the hereditary material.
Proofreading by DNA polymerase: as DNA polymerase adds nucleotides during replication, it checks each new base pair. If a mismatch is detected, the enzyme uses its 3' to 5' exonuclease activity to remove the incorrect nucleotide and replace it with the correct one.
DNA repair pathways: damage that escapes proofreading, or damage caused by environmental factors (UV light, chemicals), is handled by dedicated repair systems.
These pathways can detect and excise damaged bases, fill in gaps, and seal the repaired strand.
If repair fails, the cell may undergo apoptosis (programmed cell death) or, in some cases, accumulate mutations that contribute to cancer.
Restriction enzymes: cut DNA at specific recognition sequences, often producing sticky ends. Researchers use these to open a plasmid and insert a gene of interest.
Plasmids as vectors: a plasmid used in cloning needs three key elements:
An origin of replication so it can replicate inside the host cell.
A promoter to drive transcription of the inserted gene.
A selectable marker (e.g., antibiotic resistance or fluorescence) so researchers can tell which cells successfully took up the plasmid.
Cloning workflow: cut the plasmid with a restriction enzyme, cut the gene of interest with the same enzyme (to produce matching sticky ends), mix them, and ligate. Transform into bacteria, then select for the marker.
CRISPR-Cas9: a more recent and precise tool for genome editing.
A guide RNA is designed to be complementary to the target DNA sequence.
The guide RNA directs the Cas9 protein to the correct location in the genome.
Cas9 cuts both strands of the DNA at that site, allowing researchers to delete, replace, or insert sequences.
Restriction enzymes and plasmids are the workhorses of the biotechnology industry. Insulin, for example, is produced commercially by inserting the human insulin gene into bacterial plasmids and growing the bacteria in large fermenters.
CRISPR-Cas9 is being explored as a treatment for genetic diseases such as sickle cell anaemia, where a single base change in the haemoglobin gene causes the disease. Clinical trials have already shown promising results in correcting the mutation in patient cells.
Students often think the Hershey-Chase experiment discovered DNA. It did not discover DNA (that was Friedrich Miescher, decades earlier). It proved that DNA, rather than protein, is the molecule that carries genetic information.
"Antiparallel" does not mean the strands go in random directions. It means they run in precisely opposite orientations, 5' to 3' and 3' to 5', side by side.
Students sometimes believe that DNA polymerase only builds new strands. It also proofreads, checking each nucleotide as it is added and removing mismatches.
CRISPR-Cas9 is sometimes described as if it edits DNA on its own. The Cas9 protein cuts the DNA; the cell's own repair machinery then makes the edit, guided by whatever template the researcher provides (or, if no template is given, the break is repaired imperfectly, often disabling the gene).
The Hershey-Chase experiment is a staple exam question. Know the logic: phosphorus labels DNA, sulphur labels protein, only phosphorus enters the cell, therefore DNA is the hereditary material.
Be able to explain why the two DNA strands are antiparallel and what that means for replication (DNA polymerase can only synthesise in the 5' to 3' direction).
Expect a question on the components a plasmid needs for cloning: origin of replication, promoter, selectable marker.
Restriction enzymes and sticky ends are commonly tested. Know how matching sticky ends allow fragments from different sources to be joined.
CRISPR questions may ask you to describe the role of each component: guide RNA (targeting), Cas9 (cutting), cell repair machinery (editing).
True or False: In the Hershey-Chase experiment, radioactive sulphur was found inside the bacterial cells.
False. Only radioactive phosphorus (marking DNA) was found inside; sulphur (marking protein) stayed outside.
Fill in the blank: A always pairs with ________ and G always pairs with ________.
T (thymine) and C (cytosine).
True or False: DNA polymerase can synthesise new DNA in both the 5' to 3' and 3' to 5' directions.
False. DNA polymerase synthesises only in the 5' to 3' direction.
Fill in the blank: Restriction enzymes cut DNA at specific sequences and often produce single-stranded overhangs called ________.
Sticky ends.
True or False: CRISPR-Cas9 uses a guide RNA to locate the target sequence in the genome.
True.
Q: Describe the logic of the Hershey-Chase experiment. Why did the researchers use phosphorus and sulphur as labels?
A: DNA contains phosphorus but no sulphur. Protein contains sulphur but no phosphorus. By labelling each molecule with its unique element, they could track which one entered the host cell. Only phosphorus was found inside, proving DNA is the hereditary material.
Q: A researcher cuts a plasmid with restriction enzyme EcoRI and cuts a human gene with the same enzyme. Explain why the two fragments can be joined.
A: EcoRI produces the same sticky ends on both the plasmid and the human gene fragment. The complementary single-stranded overhangs hydrogen-bond with each other, and DNA ligase seals the phosphodiester backbone, creating a recombinant plasmid.
Q: What would happen if a plasmid lacked an origin of replication?
A: The plasmid would not be able to replicate inside the host cell. It might enter the cell, but it would be diluted out as the cell divides, and the gene of interest would not be maintained or expressed in the bacterial population.
Q: Explain how CRISPR-Cas9 targets a specific gene. What role does each component play?
A: The guide RNA is a short RNA sequence designed to be complementary to the target DNA. It binds to Cas9 and directs it to the matching site in the genome. Cas9 then cuts both strands of the DNA at that location. The cell's own repair machinery either disables the gene (via imprecise repair) or inserts a new sequence (if a repair template is provided).
DNA structure is the foundation for understanding replication, transcription, and translation. The concept of base pairing reappears in transcription (where RNA polymerase uses the template strand) and in CRISPR (where the guide RNA pairs with the target DNA). Genetic engineering tools connect to gene regulation, since researchers use inducible promoters (like the lac promoter) to control when a cloned gene is expressed.
DNA structure, double helix, base pairing, complementary base pairing, antiparallel, phosphodiester bond, purine, pyrimidine, adenine, thymine, guanine, cytosine, deoxyribose, Hershey-Chase experiment, DNA replication, DNA polymerase, proofreading, exonuclease, DNA repair, mismatch repair, restriction enzyme, sticky ends, blunt ends, plasmid, vector, cloning, selectable marker, antibiotic resistance, origin of replication, promoter, CRISPR, Cas9, guide RNA, genome editing, gene therapy, recombinant DNA, molecular cloning, BIO 311C