DNA Replication, Sequencing, and Cell Division - Study Notes 9/11/25 genetics
Replication Overview
Replication bubbles form when DNA unwinds around origins of replication, creating a bidirectional fork that extends as unwinding proceeds.
Leading strand synthesis is continuous in the $5^{\prime} \rightarrow 3^{\prime}$ direction from the origin of replication.
Represented by red arrows indicating continuous synthesis on the leading strand.
As the bubble grows, more template becomes exposed on both strands, enabling synthesis of new DNA on both templates.
Lagging strand synthesis occurs in the $5^{\prime} \rightarrow 3^{\prime}$ direction toward the origin, producing short fragments (Okazaki fragments) that are later joined.
Orientation and anchoring: the origin of replication and replication fork serve as reference points to determine directionality for both leading and lagging strands.
Termination of elongation occurs when the template strand is no longer single-stranded (in linear chromosomes).
Termination sequences block polymerase and cause polymerase to dissociate, ending replication.
In prokaryotes (circular genomes), termination occurs when replication forks meet.
In eukaryotes (linear chromosomes), replication ends at chromosome ends after fork progression reaches termini.
Prokaryotes vs Eukaryotes: Key Differences in Replication
Origins of replication
Prokaryotes (e.g., E. coli): usually a single origin per genome.
Eukaryotes: multiple origins per chromosome to handle large genome size and increase efficiency.
Replication bubbles
In bacteria: a single or few bubbles expanding until forks meet.
In eukaryotes: many bubbles operating simultaneously along chromosomes; termination when bubbles merge.
Licensing of origins (Eukaryotes)
Origins are licensed by specialized proteins during G1 phase (origin licensing complex).
DNA polymerases
Prokaryotes: a simpler set of polymerases; main replication enzyme is DNA polymerase III; DNA polymerase I replaces primers.
Eukaryotes: multiple polymerases with distinct roles (e.g., Pol α/primase for primer synthesis, Pol δ and Pol ε for lagging and leading strand synthesis, respectively).
Eukaryotes also have dedicated enzymes for primer removal and error correction beyond the bacterial system.
End Replication and Telomeres (Eukaryotes)
End replication problem in linear chromosomes: primers at ends leave a gap after removal since there is no template to extend from the RNA primer.
Consequence: progressive shortening of linear chromosomes if unresolved, potentially affecting genes if shortening reaches euchromatin regions.
Telomeres and telomerase
Telomeres are repetitive DNA sequences at chromosome ends that protect essential genes.
Telomerase RNA component is complementary to telomeric sequences and helps extend ends to compensate for primer removal.
Telomerase activity is high in proliferative cells (fetal, germline, bone marrow, and other cells with high replication demand).
Telomerase activity helps prevent loss of important genetic information during repeated cell divisions.
Typical human telomere repeat motif is $\texttt{TTAGGG}$, repeated many times; telomerase adds these repeats to extend chromosome ends.
Applied Molecular Biology: From Replication to DNA Technologies
Polymerase Chain Reaction (PCR): in vitro DNA amplification of a specific segment.
Gel electrophoresis: separates DNA fragments by size to analyze PCR products or STRs.
Sanger sequencing (Dideoxy sequencing): sequencing a single gene using chain-terminating ddNTPs in a tube.
Next-generation sequencing (NGS, high-throughput sequencing): sequencing an entire genome in a slide- or chip-based workflow, enabling parallel sequencing of many fragments.
Polymerase Chain Reaction (PCR)
Purpose: amplify a specific region of DNA to obtain lots of copies for analysis.
Primer design
Primers are short DNA oligos designed to flank the target region; specificity depends on sequence and length.
Example in transcript: a primer around ~22 bases long can uniquely identify a position in the human genome; discussions mention ~22 bases as typical length for uniqueness.
Possible combinations: with four nucleotides (A, T, C, G), a 22-base primer has $4^{22}$ possible sequences.
A well-designed primer binds to complementary template regions on both strands to define the amplification target.
Core steps of PCR cycling
Denaturation: heat DNA to separate strands, creating single-template strands.
Annealing: primers bind (anneal) to complementary sequences on the templates.
Extension: a DNA polymerase extends from the primer to synthesize new DNA; a thermostable polymerase (e.g., Taq polymerase, from Thermus aquaticus) is used to operate at high temperatures.
Repetition: cycle this process approximately 30–35 times to generate large amounts of the target DNA.
Primer extension context in the diagram: priming occurs next to the region to be replicated, mirroring the in vivo initiation of replication with RNA primers.
Output: exponential amplification; the number of copies after n cycles under ideal doubling is N = N0 \cdot 2^{n} (assuming 100% efficiency). More generally, N = N0 \cdot (1 + E)^{n} where $E$ is the amplification efficiency per cycle (0 < E ≤ 1).
Gel Electrophoresis and STRs (Forensic Relevance)
Gel electrophoresis separates PCR products by size: longer fragments migrate more slowly than shorter ones.
Short Tandem Repeats (STRs): loci with repeats of a short DNA motif; the number of repeats varies among individuals and is highly polymorphic.
Variation at a locus: the same chromosomal position can have different repeat counts on homologous chromosomes (e.g., one chromosome with 35 repeats and the homolog with 2 repeats at the same locus).
In forensic contexts, STR length polymorphisms produce distinctive banding patterns that can differentiate individuals.
Example discussion: at a given locus (e.g., chromosome 1, mid-arm region), one individual may have 35 repeats while another has 2 repeats; when PCR-amplified, these differences yield different fragment lengths that are resolved on a gel or via capillary electrophoresis.
Key concept: the same locus can have different repeat lengths on the two homologous chromosomes, giving two different alleles in an individual.
Sanger Sequencing (Dideoxy Sequencing)
Concept: sequencing DNA in a tube by incorporating chain-terminating ddNTPs alongside normal dNTPs.
Basic mechanism:
DNA polymerase requires a 3′-OH to extend; ddNTPs lack this 3′-OH and terminate DNA synthesis when incorporated.
A mixture of dNTPs and ddNTPs in each reaction leads to fragments of varying length that terminate at each possible base.
The resulting terminated fragments can be separated by size to read the sequence.
In practice:
Separate reactions are set up for each base (A, T, C, G) or use fluorescently labeled ddNTPs to read bases via a detector.
A typical setup includes four separate wells with the corresponding ddNTPs; a readout indicates the terminal base of each fragment.
Fluorescent sequencing and reversible terminator chemistry:
Modern Sanger-like methods use fluorescence to identify the terminating base in each fragment; the terminator can be reversible to allow cycling and readout.
Readout and interpretation:
The output is a chromatogram or a plotted series, where colored peaks correspond to bases; the sequence is read by the order of fragment lengths and their terminal bases.
Conceptual example described in the transcript: a single DNA segment is sequenced by repeating cycles of nucleotide incorporation and termination, with signals detected by fluorescence to determine the sequence.
Next-Generation Sequencing (NGS): High-Throughput Sequencing
Concept: sequencing many DNA fragments in parallel to read entire genomes rather than a single locus.
Workflow overview described in the transcript:
DNA is fragmented and tethered onto a slide or a surface.
A universal primer is used to initiate synthesis on all fragments in parallel.
Each cycle adds a labeled nucleotide that is detected by fluorescence; a reversible terminator allows controlled incorporation in each cycle.
After each cycle, terminators are removed, and the next cycle begins.
The platform captures images across the slide to determine which nucleotide was added at each fragment position.
Read structure and assembly:
Reads are short sequences from many fragments that overlap; overlapping reads are aligned to reconstruct the entire genome.
Coverage refers to how many times each base is read; higher coverage increases accuracy.
Practical implication: sequencing in a high-throughput, parallel fashion enables whole-genome sequencing and large-scale genomics projects.
From PCR to Genome: Conceptual Link to Cell Division
Link to cell division: accurate replication is essential so that cells can divide with faithful genetic information.
In Prokaryotes: binary fission involves replicating the genome once before division.
In Eukaryotes: the cell cycle includes S phase (DNA replication), followed by mitosis and cytokinesis.
Mitosis and Chromosome Segregation
Purpose of mitosis: produce two genetically identical daughter cells with a complete copy of the genome.
Chromosome architecture in the nucleus:
Each chromosome occupies its own territory within the nucleus; chromatin organization is not random.
Key stages and features:
Prometaphase: chromosomes begin to align; cohesins hold sister chromatids together along the length of each chromosome.
Cohesins: protein rings that encircle sister chromatids to keep them connected until separation.
Mitosis results in two halves of the cell that each receive one copy of each chromatid.
Cytokinesis:
Animal cells: division is achieved by a contractile ring that pinches the cytoplasm to form two daughter cells.
Plant cells: a cell plate forms, dividing the cytoplasm and creating two distinct daughter cells.
Timing with telophase and cytokinesis:
In many cells, cytokinesis occurs concurrently with telophase.
Foundational Connections and Real-World Relevance
Foundational principles:
The central dogma relationship between replication fidelity and genetic inheritance.
The spatial organization of chromosomes and replication dynamics play a crucial role in genome stability.
Real-world relevance of sequencing technologies:
PCR enables diagnostics, cloning, and forensic analyses.
STR analysis underpins many forensic and paternity tests due to high polymorphism at repeat loci.
Sanger sequencing remains a gold standard for confirmatory sequencing of specific loci.
NGS enables comprehensive genome sequencing, variant discovery, and large-scale genomics studies.
Ethical and practical implications:
Privacy concerns arise from genome sequencing data and STR-based profiling, which can reveal sensitive information about individuals and relatives.
Forensic use of STRs must balance evidentiary value with potential privacy issues and discriminatory outcomes.
Advances in sequencing raise considerations about data storage, sharing, and use in research and clinical settings.
Summary of Key Points (Quick Reference)
Replication mechanics:
$5^{\prime}$ to $3^{\prime}$ synthesis on the leading strand; lagging strand synthesized in short fragments toward the origin; replication bubbles expand and forks meet to terminate.
Prokaryotic vs Eukaryotic replication:
Prokaryotes: single origin; eukaryotes: multiple origins; licensing in G1; more complex polymerase system; end replication problem addressed by telomeres and telomerase.
Telomeres and telomerase:
Ends of linear chromosomes require telomerase activity to maintain genome integrity in proliferative cells.
PCR and primer design:
Design primers ~22 bases long for specificity; 30–35 cycles; denaturation, annealing, extension; output scales as N = N_0 \cdot (1 + E)^n with $E$ efficiency.
STRs and forensic applications:
Variation in repeat numbers across individuals leads to different amplicon sizes used for identification.
Sanger sequencing:
Use of ddNTPs to terminate synthesis; fluorescence readouts enable base calling; potential use of reversible terminators.
Next-generation sequencing:
Parallel sequencing of many fragments; reads overlap; assembly yields genome sequences.
Mitosis and cytokinesis:
Sister chromatids segregate to create two genetically identical daughter cells; cohesins; animal vs plant cytokinesis mechanisms.
Real-world implications:
Genomic technologies enable diagnostics, forensics, and personalized medicine, but require careful ethical and privacy considerations.