D1.1 DNA Replication

What is the purpose of DNA replication?

To produce exact copies of DNA with identical base sequences for reproduction, growth, and tissue repair.

Why is DNA replication necessary in multicellular organisms?

For growth by cell division, tissue replacement, and repair of damaged cells.

Examples of replication in reproduction?

Hydra reproduces asexually by budding; Micrasterias splits in two; gametes require replicated DNA for sexual reproduction.

What does semi-conservative replication mean?

Each new DNA molecule consists of one original (parent) strand and one newly synthesized strand.

Why is complementary base pairing important in replication?

It ensures accuracy in copying: A pairs with T, C pairs with G, conserving the genetic code.

How do hydrogen bonds stabilize DNA replication?

Complementary bases form hydrogen bonds, ensuring correct pairing and stable replication.

What is the role of helicase in DNA replication?

Helicase unwinds the DNA helix and separates the two strands by breaking hydrogen bonds using ATP.

What is PCR (polymerase chain reaction)?

An automated process that amplifies small amounts of DNA using cycles of heating and cooling.

What enzyme is used in PCR and why?

Taq polymerase, because it is heat-tolerant and survives high denaturation temperatures. Taq polymerase catalyzes the synthesis of new DNA strands by adding nucleotides to a growing chain, effectively copying a target DNA sequence

What are the three steps of PCR?

1) Denaturation: DNA sample is heated to separate it into two single strands (~95ºC for 1 min)

2) Annealing: DNA primers, which are short RNA sequences serving as starting points for polymerase, attach to the 3' ends of the target sequence ( 55ºC for 1 min)

3) Elongation: A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min) by adding nucleotides.

What is the result of 30 PCR cycles?

Over 1 billion copies of the target DNA sequence.

What is gel electrophoresis?

A method of separating DNA fragments by size through a gel under an electric field.

Why does DNA move in electrophoresis?

Because the sugar-phosphate backbone is negatively charged, DNA migrates towards the positive electrode.

How do DNA fragments separate in a gel?

Smaller fragments move further through the gel mesh; larger fragments travel shorter distances.

What are STRs?

Short tandem repeats, sequences of 2-6 nucleotides repeated many times, highly variable between individuals.

What are applications of PCR + gel electrophoresis?

Crime scene investigations, paternity testing, diagnosing diseases (e.g., coronaviruses).

How is DNA profiling used in forensics?

DNA bands from a suspect are compared to crime scene DNA; close matches indicate identity.

How is DNA profiling used in paternity testing?

Child's DNA bands are compared with mother's and potential father's; shared bands indicate parentage.

Why is helicase essential?

Without helicase, the DNA strands would remain wound and inaccessible for replication.

Why is Taq polymerase used in PCR?

Because it originates from Thermus aquaticus, a heat-tolerant extremophile bacterium.

Why do shorter DNA fragments travel further in electrophoresis?

They encounter less resistance in the gel matrix.

Why are STRs useful in DNA profiling?

They are highly variable, making each person's DNA fingerprint unique.

What marks the 5' end of a nucleotide?

The phosphate group attached to carbon 5 of deoxyribose.

What marks the 3' end of a nucleotide?

The hydroxyl (OH) group attached to carbon 3 of deoxyribose.

Where does the energy for nucleotide attachment come from?

From hydrolysis of two phosphates from the incoming nucleoside triphosphate.

How many RNA primers are needed on the leading strand?

Only one, because replication is continuous.

What is the role of single-stranded binding proteins?

Keep separated strands apart so nucleotides can bind.

What is the role of RNA primase?

Synthesizes short RNA primers to provide attachment points for DNA polymerase III.

What is the role of DNA polymerase III?

Adds deoxynucleoside triphosphates to the 3' end, synthesizing in the 5' → 3' direction.

What is the role of DNA polymerase I?

Removes RNA primers and replaces them with DNA nucleotides.

How does DNA polymerase III proofread?

It removes mismatched nucleotides at the 3' end and replaces them with correctly paired bases.

How does replication proceed from origins?

Bidirectionally on both strands.

Why does replication proceed only 5' → 3'?

Because nucleotides can only be added to the free 3' OH group of the previous nucleotide.