Human Genetics 27

DNA is structured as a double helix composed of two strands.
The two strands are held together by hydrogen bonds between the nitrogenous bases.
To replicate, the double helix must be unwound, breaking these hydrogen bonds to separate the strands.

Watson and Crick proposed that DNA replication is semiconservative. - This means that each new double helix contains one original strand (parental) and one newly synthesized strand.
After replication, two identical double helices are formed from each original parental double helix. - The original strand (blue) serves as a template for a new strand (red).

Experiment aimed to prove the semiconservative model of DNA replication.
Methodology: - Labeled old and new DNA using different isotopes of nitrogen (heavy and light). - Centrifuged DNA samples after replication.
Results: - Dense N-15 (heavy nitrogen) was incorporated into the original strands, while N-14 (light nitrogen) was incorporated into newly synthesized strands. - Each daughter DNA molecule contains one heavy (old) strand and one light (new) strand, consistent with semiconservative replication.

Requirements for DNA replication: - Template DNA - DNA polymerase enzyme - Monomers (nucleotides) - Additional enzymes for the replication process

The parental DNA strands unwind and separate at origins of replication across the chromosome, allowing multiple replication bubbles to form simultaneously.
Each parental strand serves as a template for complementary base pairing: - Adenine (A) pairs with Thymine (T) - Guanine (G) pairs with Cytosine (C)

DNA polymerase synthesizes new DNA strands by adding nucleotides complementary to the template strands.
Hydrogen bonding occurs between newly added nucleotides and the template strand bases.
Semiconservative nature: New DNA consists of one original (parental) strand and one new strand.

The site where DNA is actively unwound is termed the replication fork.
Characteristics of the replication fork: - Tines are made up of new and old strands. - Areas that are unwinding exhibit breaking of hydrogen bonds to allow strand separation.

Helicase:
- Unwinds the DNA double helix and breaks hydrogen bonds, separating the original strands.
Single-Stranded Binding Proteins (SSBs): - Bind to single-stranded DNA to prevent reformation of the double helix and stabilize separated strands.
Primase: - An RNA polymerase that adds a short RNA primer to the template strand, providing a 3'-OH group for DNA polymerase.
DNA Polymerase: - Binds to the 3'-OH end of the primer and begins synthesizing complementary DNA sequences. - It works in a 5' to 3' direction, continuously adds nucleotides one by one from the nucleotide pool.
DNA Ligase: - Joins Okazaki fragments on the lagging strand by sealing the nicks in the sugar-phosphate backbone, forming phosphodiester bonds. - Facilitates removal of RNA primers and replaces them with DNA nucleotides.

Leading Strand: - Synthesis proceeds in the same direction as the replication fork movement (continuously). - Copied in a 5' to 3' direction.
Lagging Strand: - Copied in the opposite direction relative to the replication fork, leading to the formation of Okazaki fragments (discontinuously). - Each fragment must be separately synthesized and later joined by DNA ligase.

DNA polymerase has proofreading abilities to correct errors during nucleotide addition. - Can backtrack to remove incorrectly paired nucleotides and replace them with the correct ones.
Some mistakes are retained and may lead to mutations, which can contribute to cancer or evolutionary changes.

A laboratory technique used to amplify specific DNA sequences exponentially.
Utilizing principles of DNA replication, it allows for the creation of billion copies from a small starting sample.
Steps of PCR: 1. Selection of Target DNA Sequence: Choose specific DNA sequence to amplify. 2. Design of Primers: Create primers complementary to the ends of the target sequence. 3. Addition of Nucleotides and DNA Polymerase: Mix with free nucleotides and a heat-stable DNA polymerase. 4. Thermal Cycling: - Denaturation: Heat DNA to separate strands. - Annealing: Cool to allow primers to bind to the target sequence. - Extension: Warm slightly for polymerase to synthesize new DNA strands from the primers. 5. Repeat Cycles: Exponential amplification achieved through repeated cycling of the thermal steps.

Used in diagnostics (e.g., detecting viral infections), forensic analysis, ecological research, and ancient DNA studies.
Enabled rapid testing methods for diseases like COVID-19.

Traditional Sequencing - Sanger Sequencing: - Developed by Frederick Sanger, used to sequence DNA by incorporating chain-terminating nucleotides.
Next Generation Sequencing (NGS): - Fast, automated, and capable of sequencing genomes in hours. - Differences from Sanger Sequencing: - Relies on massively parallel processing of multiple reactions simultaneously. - Fluorescent labeling of nucleotides for identification, eliminating the need for gel-based separation.

Describes the flow of genetic information: - DNA → RNA → Protein - Gene expression involves transcription of DNA to RNA, which is then translated into proteins.
Transcription: Conversion of DNA information into RNA.
Translation: Formation of polypeptides from RNA using ribosomes and tRNA.
Importance of transcription as an intermediary: - Enables regulation of gene expression and acts as a means of transporting genetic information from the nucleus to the cytoplasm for protein synthesis.