Chapter 6: DNA and Biotechnology

A nucleotide has three parts: a nitrogenous base, a five-carbon sugar, and a phosphate (or phosphates). Add the phosphate and it becomes a nucleoTIDE. Remove the phosphate and it is just a nucleoSIDE (sugar + base).

The Five Bases

Two families of bases, distinguished by their ring structure:

• Purines: two fused rings. Adenine (A) and guanine (G).
• Pyrimidines: one ring. Cytosine (C), thymine (T), and uracil (U).

Thymine is in DNA only. Uracil is in RNA only. A, G, and C are in both.

Sugars: DNA vs. RNA

• DNA uses 2-deoxyribose - C2 has no hydroxyl (just -H).
• RNA uses ribose - C2 has a hydroxyl (-OH).

That single hydroxyl difference makes RNA far less chemically stable than DNA. The 2’-OH of RNA can attack the phosphodiester backbone intramolecularly, cleaving the chain. DNA, lacking this attacker, is much more stable - appropriate for long-term information storage.

Nucleoside vs. Nucleotide Naming

The Phosphodiester Backbone

Nucleotides polymerize through phosphodiester bonds: the 5’-phosphate of one nucleotide attaches to the 3’-OH of the next. This gives the backbone its directionality. Every DNA or RNA strand has a 5’ end (free phosphate) and a 3’ end (free -OH).

Polymerases always add new nucleotides to the 3’-OH, so synthesis runs 5’ → 3’. Reading and writing conventions follow this: a sequence written as ATCG means the 5’ end has A and the 3’ end has G.

DNA is a right-handed double helix of two antiparallel polynucleotide strands. The backbones run on the outside; the bases pair in the center.

Base Pairing

The two strands are held together by hydrogen bonds between paired bases. Watson-Crick base pairing follows strict rules:

• A pairs with T via 2 hydrogen bonds.
• G pairs with C via 3 hydrogen bonds.

A purine always pairs with a pyrimidine, keeping the helix diameter constant.

Antiparallel Strands

The two strands run in opposite directions. One strand is 5’ → 3’ top to bottom; the other is 5’ → 3’ bottom to top. Each base on one strand pairs with the complementary base directly across from it on the other strand.

This antiparallel arrangement has a major consequence: since DNA polymerases only synthesize 5’ → 3’, the two strands at a replication fork must be copied differently. One is made continuously (leading strand), one is made in fragments (lagging strand).

Grooves

The helix has two grooves where proteins can read the base sequence without unwinding the helix:

• Major groove: wider; exposes more of the base edges. Most transcription factors recognize DNA sequences here.
• Minor groove: narrower; less information. Some specialized proteins read sequences here.

Chargaff’s Rules

Before Watson and Crick, Erwin Chargaff showed that for any double-stranded DNA sample:

1. The amount of A equals the amount of T, and the amount of G equals the amount of C.
2. The percent of purines (A + G) equals the percent of pyrimidines (T + C), which is always 50%.

These relationships make sense only if A pairs with T and G pairs with C. Chargaff’s data was a critical clue in Watson and Crick’s 1953 model.

DNA Denaturation, Reannealing, and Hybridization

The hydrogen bonds between paired bases are much weaker than the covalent backbone, so you can pull the two strands apart without breaking the sequence. This is denaturation (sometimes called “melting”). Heat is the usual cause in the lab; extreme pH or chaotropes (urea, formamide) also work.

• Melting temperature (Tm): the temperature at which half of the DNA is single-stranded and half is double-stranded. Higher GC content = higher Tm (three H-bonds per GC vs. two per AT). Longer DNA and higher salt also raise Tm.
• Reannealing: if cooling is slow after denaturation, the separated strands will find their complements again and reform the double helix. The optimal reannealing temperature is about 20-25°C below Tm.
• Hybridization: single-stranded DNA (or RNA) from one source pairs with complementary single-stranded nucleic acid from another source. This is how probes work in Southern and Northern blots, how primers find their targets in PCR, and how microarrays read gene expression.

Chromatin Structure in the Nucleus

A single human cell contains about 2 meters of DNA packaged into a nucleus only a few microns across. The packaging solution is chromatin - DNA wound around proteins and folded hierarchically.

• Histones: small basic proteins rich in lysine and arginine (positively charged to bind the negative DNA backbone). Five histone types: H2A, H2B, H3, H4, and the linker H1.
• Nucleosome: ~147 bp of DNA wrapped about 1.65 turns around a histone octamer (two copies each of H2A, H2B, H3, H4). Nucleosomes are the fundamental repeating unit of chromatin, often described as “beads on a string.”
• 30-nm fiber: nucleosomes coil into thicker fibers, with H1 stabilizing the coil.
• Higher-order folding produces the condensed metaphase chromosome, which is ~10,000-fold shorter than the naked DNA.

Euchromatin vs. Heterochromatin

• Euchromatin: loosely packed, transcriptionally active. Appears lighter under microscopy.
• Heterochromatin: densely packed, transcriptionally silent. Appears darker. Constitutive heterochromatin (always condensed, like centromeres and telomeres) vs. facultative heterochromatin (tissue- or time-specific silencing, like the inactivated X chromosome).

Telomeres and Centromeres

• Telomeres: repetitive sequences (TTAGGG in humans) at chromosome ends. They cap the ends to prevent fusion and degradation, and they shorten with each round of replication unless telomerase extends them. Shortening is linked to cellular aging; telomerase reactivation is a hallmark of many cancers.
• Centromeres: heterochromatic repetitive regions in the middle of each chromosome that anchor sister chromatids together and serve as the assembly site for the kinetochore during mitosis.

Single-Copy vs. Repetitive DNA

Only about 1-2 percent of the human genome codes for protein. The rest includes regulatory elements, introns, and large stretches of repetitive DNA.

• Single-copy DNA: most protein-coding genes. Present as one (or a few) copies per genome.
• Repetitive DNA: tandem repeats (like centromeric and telomeric repeats) and interspersed repeats (like transposon-derived SINEs and LINEs). Some repetitive regions are structural; others are evolutionary relics.

DNA replication produces two identical double helices from one original. The key experiment (Meselson-Stahl, 1958) showed that replication is semiconservative - each daughter molecule contains one original strand and one newly synthesized strand.

The Replication Fork

Replication begins at an origin of replication. The double helix is unwound, creating a Y-shaped replication fork. DNA polymerase synthesizes new DNA on both template strands - but each strand is made differently because of the antiparallel geometry.

• Leading strand: synthesized continuously 5’ to 3’ toward the fork.
• Lagging strand: synthesized discontinuously in short 5’ to 3’ fragments called Okazaki fragments. These are later joined together.

Key Enzymes

| Enzyme | Job |
|--------|-----|
| Helicase | Unwinds the double helix at the fork |
| Topoisomerase (gyrase in bacteria) | Relieves supercoiling ahead of the fork |
| Single-strand binding proteins (SSBs) | Prevent reannealing of the separated strands |
| Primase | Synthesizes short RNA primers (~10 nt) that DNA polymerase can extend |
| DNA polymerase III (bacteria) / δ, ε (eukaryotes) | Main replicative polymerase. Adds dNTPs to the 3’-OH of the growing strand |
| DNA polymerase I (bacteria) | Removes RNA primers, fills in gaps with DNA |
| DNA ligase | Seals nicks between Okazaki fragments |

Fidelity

DNA polymerases have very low error rates - about one mistake per $10^{7}$ bases. Several mechanisms contribute:

• Base selection: the active site is shaped to fit correct base pairs much better than mismatches.
• Proofreading: most replicative polymerases have 3’ to 5’ exonuclease activity that removes a mispaired base just after it is added, then tries again.
• Mismatch repair: post-replication, a separate system scans newly made DNA for any surviving mismatches and corrects them (see DNA repair section).

Together these systems bring the overall error rate down to about one mistake per $10^{9}$ bases.

DNA gets damaged constantly. UV light, chemical mutagens, reactive oxygen species, and spontaneous hydrolysis all cause lesions. Without repair, mutations would accumulate rapidly. Several distinct repair pathways exist, each specialized for a different kind of damage.

Proofreading

Part of replication itself. Most DNA polymerases have a 3’ to 5’ exonuclease activity that reads back after placing a new nucleotide. If the new base is not correctly paired, the polymerase chops it off and tries again. This catches most errors during synthesis.

Mismatch Repair (MMR)

Fixes the rare errors that escape proofreading. The mismatch repair system scans freshly replicated DNA, recognizes mispaired bases, and replaces the incorrect nucleotide on the newly synthesized strand (not the template). How does it know which strand is new? In bacteria, the parental strand is methylated at certain sequences and the new strand is not yet methylated. In eukaryotes, the mechanism is less clear but probably uses nicks in the new strand.

MMR defects cause hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome) - mutations in MLH1, MSH2, and other MMR genes.

Base Excision Repair (BER)

Repairs single damaged bases (like oxidized or deaminated ones). A DNA glycosylase removes the damaged base, leaving an abasic site. An endonuclease nicks the backbone. A polymerase fills the gap with a correct nucleotide, and ligase seals the nick.

Nucleotide Excision Repair (NER)

Repairs bulky lesions that distort the helix, like UV-induced thymine dimers. Endonucleases cut out a short segment (~12-24 nucleotides) around the damage. Polymerase fills the gap, ligase seals it.

NER defects cause xeroderma pigmentosum (XP) - patients cannot repair UV damage, develop skin cancers as children, and must avoid sunlight entirely.

Double-Strand Break Repair

Double-strand breaks are the worst kind of damage - both strands are cut. Two repair paths:

• Homologous recombination (HR): uses an identical sister chromatid as a template. Accurate but requires an intact copy, so it is restricted to the S and G2 phases of the cell cycle. BRCA1 and BRCA2 proteins are essential components. Mutations in BRCA$\frac{1}{2}$ cause hereditary breast and ovarian cancer.
• Non-homologous end joining (NHEJ): directly glues the two ends back together without a template. Fast but error-prone - small insertions or deletions often result. Used throughout the cell cycle.

Clinical Examples

Restriction enzymes are bacterial proteins that cut DNA at specific sequences. Bacteria evolved them to slice up invading viral DNA. Molecular biologists hijack them to cut and paste DNA in the lab - the foundation of modern genetic engineering.

Recognition Sequences

Restriction enzymes recognize and cut palindromic sequences. A palindrome in DNA reads the same 5’ to 3’ on both strands. For example, EcoRI recognizes:

5'-G A A T T C-3'
3'-C T T A A G-5'

Read the top strand 5’ to 3’ (GAATTC), then read the bottom strand 5’ to 3’ (from right to left on the page, GAATTC). Same sequence. That is a palindrome.

Sticky Ends and Blunt Ends

EcoRI cuts between G and A on each strand, leaving overhangs:

5'-G            A A T T C-3'
3'-C T T A A            G-5'

These single-stranded overhangs (sticky ends) can base-pair with any other EcoRI-cut DNA. This is what makes cloning work - you cut a gene of interest with EcoRI, cut your plasmid vector with EcoRI, and the sticky ends recombine. Ligase seals the nicks.

Some enzymes (like EcoRV, SmaI) cut straight across, producing blunt ends. Blunt ends are less selective in ligation but still usable.

Plasmid Vectors

A plasmid is a small circular DNA molecule found naturally in bacteria. Cloning vectors are engineered plasmids with:

• An origin of replication (ori) so the plasmid replicates in bacterial hosts.
• A multiple cloning site (MCS) - a stretch of unique restriction sites where you can insert your gene.
• A selectable marker (usually an antibiotic resistance gene) so bacteria that contain the plasmid can be selected.

To clone a gene:

1. Cut both the gene and the plasmid with the same restriction enzyme.
2. Mix fragments with DNA ligase.
3. Transform bacteria with the ligated product (e.g., by heat shock or electroporation).
4. Grow bacteria on a selective medium (with antibiotic) - only transformed bacteria with the plasmid survive.
5. Confirmed bacterial colonies now carry millions of copies of your insert.

Applications

Recombinant DNA technology enables:

• Protein production: human insulin, growth hormone, and many other therapeutics are made by bacteria engineered to express the human gene.
• Gene analysis: any gene can be cloned and studied - its sequence, expression, and function.
• Genetic engineering: cloning plus modern tools (CRISPR, transgenic organisms) has built modern biotechnology.

PCR is a molecular photocopier. Starting from one DNA molecule, PCR can produce billions of copies in a few hours. It is the foundation of nearly every modern DNA technique: forensics, diagnostics, cloning, sequencing, genetic testing.

The Cycle

Each PCR cycle has three temperature steps:

1. Denaturation (~95°C): heat separates the two DNA strands.
2. Annealing (~50-65°C): temperature drops, allowing short primers to bind their complementary sequences on each strand.
3. Extension (~72°C): heat-stable DNA polymerase (Taq) extends each primer 5’ to 3’, doubling the target region.

Repeat 25-35 times. One starting molecule becomes 2, then 4, then 8, and so on. After 30 cycles, you have approximately $2^{30}$ = about 1 billion copies.

Key Ingredients

• Template DNA: the starting material (one molecule is enough).
• Primers: two short (~20 nt) DNA oligonucleotides that flank the target region. One primer matches the top strand; the other, the bottom strand.
• dNTPs: the four nucleotide substrates (dATP, dTTP, dGTP, dCTP).
• Taq polymerase: a heat-stable DNA polymerase from the bacterium Thermus aquaticus. It survives 95°C without denaturing, so it does not have to be replaced each cycle.
• Buffer + Mg2+: enzymatic cofactors.
• Thermal cycler: machine that programs the temperature changes.

Specificity from Primers

PCR’s specificity comes from the primers. Only the region between the two primer binding sites is amplified. If you want to amplify exon 3 of a specific gene, you design primers that flank exon 3. Any other DNA in the sample is ignored because no primers bind there.

Variants

• RT-PCR (reverse transcription PCR): starts with RNA. A reverse transcriptase first converts RNA to complementary DNA (cDNA); then standard PCR amplifies the cDNA. Used to measure gene expression.
• qPCR (quantitative PCR): real-time PCR with a fluorescent probe that increases signal as amplification proceeds. Allows quantification of starting template. Used heavily in COVID testing.
• Multiplex PCR: multiple primer pairs in one reaction to amplify several targets simultaneously.

Gel electrophoresis is the standard way to separate DNA fragments by size. The DNA sample is loaded into wells at one end of a porous gel (usually agarose). An electric field drives the DNA through the gel. Small fragments travel quickly; large fragments travel slowly.

How It Works

DNA has a uniformly negative charge from its phosphate backbone. In an electric field, it moves toward the positive electrode (anode). The gel matrix has tiny pores that slow fragments based on size:

• Small fragments: slip through easily → travel far.
• Large fragments: get caught in the mesh → travel less distance.

After the run, fragments are visualized by adding a DNA-binding dye:

• Ethidium bromide: intercalates between base pairs and fluoresces under UV light.
• SYBR Safe and other modern dyes: less toxic alternatives.

The pattern of bands along each lane is the “gel image” - a visual sorting of the fragments by size.

Reading a Gel

A typical gel image has:

• A ladder lane: DNA fragments of known sizes (e.g., 100, 200, 300, 500, 1000, 2000 bp). This is the ruler.
• Sample lanes: your DNA samples. Compare bands to the ladder to estimate each fragment’s size.
• The wells are at the top (or negative end). Bands near the top are large; bands near the bottom are small.

Agarose vs. Polyacrylamide

Two gel materials are common:

• Agarose: best for DNA fragments 100 bp to 50+ kb. Large pores. Lower resolution.
• Polyacrylamide: best for small DNA (<500 bp) and for proteins. Smaller pores, higher resolution. Proteins on polyacrylamide (SDS-PAGE) were covered in Chapter 3.

The choice depends on the size range you care about. For routine cloning checks (1-5 kb plasmid fragments), agarose is standard.

A gel tells you how big your fragments are but not which one is the specific molecule you care about. Blotting adds specificity: transfer the gel to a membrane, then probe the membrane with a labeled molecule that binds only your target.

Three main blots, each for a different target molecule type.

The SNoW DRoP Mnemonic

Southern Blot (DNA)

Workflow:

1. Cut genomic DNA with a restriction enzyme.
2. Run on a gel to separate fragments by size.
3. Transfer the DNA onto a membrane (nitrocellulose or nylon).
4. Incubate the membrane with a labeled DNA probe (radioactive or fluorescent) complementary to the target sequence.
5. Wash off unbound probe, then detect where the probe stuck.

A single band on the resulting film tells you the target sequence is present in the genome and at what size the restriction enzyme cut it. Southern blotting was historically used to detect gene rearrangements, copy number variations, and specific mutations. It has largely been replaced by PCR and sequencing.

Northern Blot (RNA)

Essentially the same procedure, but starting with RNA. Used to detect and size a specific mRNA. Tells you whether a gene is expressed in a tissue, whether alternative splicing produces multiple transcript sizes, and whether the transcript is degraded. Like Southern, it has been largely supplanted by quantitative methods (qPCR, RNA-seq).

Western Blot (Protein)

Workflow:

1. Run proteins on SDS-PAGE.
2. Transfer to a membrane.
3. Probe with a specific antibody against the target protein.
4. A secondary antibody (attached to an enzyme or fluorophore) detects the primary antibody.
5. A chemiluminescent substrate reveals where the primary antibody bound.

Western blotting remains the gold standard for confirming that a specific protein is present (and at what size) in a sample. It is used throughout molecular and clinical biology.

Hybridization Probes vs. Antibodies

The key difference between Southern/Northern and Western blots:

• Southern and Northern use complementary nucleic acid probes (DNA or RNA) that base-pair with the target. Specificity comes from sequence complementarity.
• Western uses antibodies that recognize a specific protein epitope. Specificity comes from the protein-protein interaction.

DNA sequencing tells you the exact order of nucleotides. The MCAT focuses on Sanger sequencing (the classical chain-termination method) and recognizes that next-generation sequencing (NGS) is the modern high-throughput approach.

Sanger (Chain-Termination) Sequencing

Developed by Frederick Sanger in 1977. The reaction is like a PCR, with one important twist: the reaction mix contains a small amount of dideoxynucleotides (ddNTPs) in addition to the normal dNTPs.

A ddNTP lacks a 3’-OH. When DNA polymerase incorporates a ddNTP, no further nucleotides can be added - the chain terminates at that base.

Workflow

1. Mix template DNA, primer, DNA polymerase, dNTPs, and a small amount of each ddNTP (each labeled with a different fluorescent color: ddA = green, ddT = red, etc.).
2. The polymerase extends the primer using dNTPs. Occasionally it incorporates a ddNTP by accident and stops.
3. The result is a mixture of fragments of every possible length, each ending in a known fluorescent ddNTP.
4. Run the mixture through capillary electrophoresis (size-separation by size).
5. A detector reads the fluorescent color of each fragment as it passes. The order of colors = the sequence of bases.

The Chromatogram

The output of a Sanger sequencer is a chromatogram: four colored traces (one per base) with peaks at each position of the sequence. Reading the tallest peak at each position left to right gives the DNA sequence.

Next-Generation Sequencing

Modern NGS platforms (Illumina, PacBio, Nanopore) sequence billions of DNA fragments in parallel. Each platform uses different chemistry, but the general idea is the same: fragment the DNA, attach adapters, amplify, then read out each fragment in parallel. This is how the Human Genome Project went from 13 years and $3 billion (Sanger-based) to 1-2 days and $1000 (modern NGS) for a whole human genome.

You do not need to know specific NGS chemistries for the MCAT. But recognize that modern sequencing is massively parallel, not Sanger’s sequential approach.

CRISPR-Cas9 is a programmable DNA-cutting system that researchers have adapted from bacterial immunity. It is the current gold standard for gene editing because it is simple, cheap, and very precise. The 2020 Nobel Prize in Chemistry recognized the development of CRISPR as a gene-editing tool.

The Bacterial Origin

In bacteria, CRISPR (clustered regularly interspaced short palindromic repeats) is an immune system against viruses. Bacteria store short snippets of past viral DNA in a special genomic region. When the virus attacks again, the bacterium transcribes these snippets into guide RNAs that direct a Cas nuclease to cut matching viral DNA.

Molecular biologists hijacked this system. By designing a guide RNA that targets any sequence of interest, Cas9 can be directed to cut any gene in any organism.

The Two Components

1. Cas9: an RNA-guided endonuclease that cuts both DNA strands.
2. Guide RNA (gRNA): a ~100-nucleotide RNA with a 20-nucleotide spacer that base-pairs with the target DNA sequence. The rest of the gRNA binds Cas9.

Target DNA must have a PAM sequence (typically NGG for the common SpCas9) immediately adjacent. The PAM is read by Cas9 and is not part of the gRNA pairing; it prevents Cas9 from cutting bacterial CRISPR arrays (which lack PAMs).

Editing Outcomes

Cas9 makes a blunt double-strand break (DSB) ~3 bp upstream of the PAM. The cell must repair this break. The repair pathway determines the editing outcome:

• Non-homologous end joining (NHEJ): the default pathway. The two broken ends are glued back together, often with small insertions or deletions (indels). A frame-shift indel in a coding region typically knocks out the gene. This is how CRISPR produces gene knockouts.
• Homology-directed repair (HDR): if a donor DNA template with homologous ends is provided, the cell can use it to repair the break precisely. This enables knock-in of specific edits, corrections of disease mutations, or insertion of tagged sequences.

Applications

• Research: creating knockout cell lines and animals. CRISPR has replaced older gene targeting methods almost entirely.
• Agriculture: drought-resistant crops, disease-resistant livestock.
• Therapeutics: the first FDA-approved CRISPR therapy, Casgevy (2023), treats sickle cell disease by editing hematopoietic stem cells. Several cancer and genetic disease trials are ongoing.
• Diagnostics: CRISPR-based detection systems (SHERLOCK, DETECTR) use Cas13 or Cas12 to detect specific nucleic acid sequences from pathogens.