DNA Replication and Molecular Genetics Techniques

Structure of DNA

The double-helix allows strand separation, and complementary base pairing (A-T, G-C) ensures accurate copying.

Meselson-Stahl experiment goal

To determine how DNA replicates.

Meselson-Stahl experiment demonstration

DNA replicates semi-conservatively, with each new strand containing one old and one new strand.

Conservative replication results

One heavy band and one light band after the first replication.

Dispersive replication results

A single intermediate band that gradually lightens over generations.

Function of helicase

Unwinds the double helix by breaking hydrogen bonds.

Enzyme relieving strain ahead of replication fork

Topoisomerase.

Stabilization of single DNA strands

Single-strand binding proteins.

Role of primase

Synthesizes RNA primers.

Role of DNA polymerase III

Adds DNA nucleotides to the 3' end of the primer.

Role of DNA polymerase I

Removes RNA primers and replaces them with DNA.

Function of DNA ligase

Seals nicks between Okazaki fragments by forming phosphodiester bonds.

Leading vs. lagging strand

The leading strand is synthesized continuously; the lagging strand is synthesized in Okazaki fragments.

DNA polymerases and proofreading

They can reverse to fix mismatched base pairs.

Mismatch repair process

Enzymes detect distortions, remove the mismatch, DNA polymerase replaces it, and ligase seals the gap.

Consequences of DNA replication errors

Mutations that can cause cancer, genetic diseases, or evolutionary change.

Telomere shortening cause

Gaps at the 5' end of the lagging strand that cannot be filled.

Circular DNA and telomere shortening

Because circular DNA has no ends; replication can go around the circle.

Purpose of genetic screens

To discover gene function by linking mutations (genotype) to traits (phenotype).

Forward genetics

Starting with a phenotype and identifying the gene responsible.

Reverse genetics

Starting with a gene, mutating it, and observing the resulting phenotype.

Sanger sequencing

Sequences DNA using chain-terminating nucleotides for base-by-base reading.

Next-gen sequencing

Sequencing millions of DNA or RNA fragments for genome or transcriptome analysis.

PCR

Amplifying specific DNA segments using primers and DNA polymerase.

FISH

Reveals where and how much a gene is expressed by binding fluorescent probes to mRNA in cells.

RT-PCR

Measuring gene expression by converting RNA to cDNA and amplifying it.

RNA-seq

Analyzing expression levels of all genes by sequencing cDNA from mRNA.

RNA interference (RNAi)

Silences genes by degrading specific mRNA molecules.

CRISPR

Gene editing by using Cas9 and guide RNA to cut and modify specific DNA sequences.