D1.1 DNA replication

DNA replication produces exact copies of DNA with identical base sequences, apart from rare copying errors.

Accurate copies preserve genetic information when cells or organisms reproduce. In multicellular organisms, replication supplies genomes for cell division during growth and replacement of damaged or worn tissues.

Original DNA sequence → replication → two matching DNA molecules → genetic continuity in reproduction, growth and tissue replacement.

Before a skin cell divides to replace lost tissue, its DNA is copied so both daughter cells can inherit the same base sequence.

Replication copies DNA; transcription makes RNA and translation makes polypeptide. ‘Identical’ describes base-sequence information, not two newly synthesized strands without templates.

Semi-conservative replication produces DNA molecules in which each double helix contains one parental strand and one newly synthesized strand.

When the original strands separate, each acts as a template. Complementary base pairing preserves information while retaining one physical strand from the original molecule in each product.

Identify a product by checking: one old strand; one new strand; complementary pairing between them.

After one round, heavy parental DNA in a density experiment is replaced by two intermediate molecules, each containing one old and one new strand.

Semi-conservative does not mean half the bases are copied randomly; the strand pattern is the key prediction.

Helicase separates the two DNA strands, while DNA polymerase builds complementary DNA strands from the exposed templates.

Helicase unwinds the double helix and breaks hydrogen bonds between complementary bases. DNA polymerase selects complementary DNA nucleotides and joins them into a growing strand.

Helicase: unwind and break inter-strand hydrogen bonds. DNA polymerase: use each original strand as a template and join complementary nucleotides.

At a replication fork, helicase exposes template bases; polymerase then places A opposite T and C opposite G while extending the new DNA.

Helicase does not synthesize DNA, and polymerase does not separate the original strands. Detailed 5′/3′ directionality belongs to the later HL objective.

PCR amplifies a selected DNA region; gel electrophoresis then separates DNA fragments mainly by length.

Primers define the target ends. Each thermal cycle uses high temperature to separate strands, lower temperature for primer binding, and a suitable extension temperature for heat-stable Taq DNA polymerase to synthesize new DNA.

Load amplified fragments into wells. Negatively charged DNA moves toward the positive electrode through the gel; shorter fragments move farther than longer fragments in the same time.

Primers amplify a variable DNA locus; electrophoresis separates the resulting fragments, and a size marker allows their approximate lengths to be compared.

PCR increases the amount of target DNA; electrophoresis separates fragments. Primer specificity, contamination controls and the size marker affect interpretation.

PCR and gel electrophoresis support DNA profiling by amplifying and comparing variable DNA markers.

In a paternity investigation, a child's marker alleles must be explainable by the biological parents. In a forensic investigation, a crime-scene DNA pattern can be compared with reference samples.

Use several independent markers: each additional matching marker reduces the probability that an unrelated person shares the full pattern by chance. Include positive/negative controls and guard against contamination.

A child's allele not supplied by the known parent must match the candidate parent's allele at each tested marker; one matching marker is weak, whereas a consistent multi-marker pattern is stronger evidence.

A profile supports or excludes a biological relationship/source; it does not alone prove when or how DNA reached a location. More markers reduce false-match probability but do not make laboratory error impossible.

DNA strands have chemically different 5′ and 3′ ends, and DNA polymerase synthesizes only in the 5′→3′ direction.

The 5′ end is associated with the phosphate on the sugar's 5′ carbon, while the 3′ end has a free hydroxyl on the 3′ carbon. Polymerase attaches the 5′ phosphate of an incoming DNA nucleotide to the free 3′ end of the growing strand.

New nucleotide's 5′ end + growing strand's 3′ end → phosphodiester bond. Therefore extension occurs only at 3′ and the new strand lengthens 5′→3′.

A polymerase moving along a template 3′→5′ builds the complementary strand 5′→3′, adding every next nucleotide at the new strand's 3′ end.

5′→3′ describes the direction of new-strand synthesis. Polymerase cannot add to the growing strand's 5′ end.

At each replication fork, the leading strand is synthesized continuously and the lagging strand discontinuously as Okazaki fragments.

DNA polymerase can extend only 5′→3′ while the templates are antiparallel. The leading template supports synthesis toward the fork; the lagging template requires repeated synthesis away from the fork as more DNA is exposed.

Leading strand: one RNA primer, then continuous synthesis. Lagging strand: repeated RNA primers, discontinuous Okazaki-fragment synthesis, followed by primer replacement and fragment joining.

As helicase advances, one polymerase follows the fork continuously; on the other template, each newly exposed section receives another primer and becomes a separate Okazaki fragment.

Both new strands are synthesized 5′→3′. Leading/lagging refer to synthesis pattern relative to fork movement, not gene content or biological importance.

In prokaryotic DNA replication, primase, DNA polymerase III, DNA polymerase I and DNA ligase perform distinct sequential jobs.

On the lagging strand, primase repeatedly starts fragments, polymerase III extends them, polymerase I replaces each primer, and ligase seals the final backbone gaps.

This named-enzyme sequence is limited to the prokaryotic system. Polymerase I replaces primers; ligase does not synthesize the missing DNA nucleotides.

DNA polymerase III proofreads a newly added base at the growing strand's 3′ terminal and corrects a mismatch before replication continues.

A non-complementary base pair distorts the new DNA. Polymerase III removes the mismatched terminal nucleotide, exposes the 3′ end again and inserts a nucleotide complementary to the template.

Mismatch at 3′ terminal → polymerase III detects it → incorrect nucleotide removed → correct complementary nucleotide added → 5′→3′ extension resumes.

If an incorrect nucleotide is added opposite a template G, proofreading removes it from the 3′ end and replaces it with C before the strand is extended further.

Proofreading greatly improves accuracy but does not eliminate every mutation. This objective is specifically polymerase III correction of a mismatched 3′ terminal.