DNA Structure: Nucleotides

• DNA's building blocks are nucleotides.
• Nucleotides consist of a nitrogenous base, 5-carbon sugar (pentose), and a phosphate group.
• Nucleotides are named according to their nitrogenous base.
• Nitrogenous bases include the purines adenine (A) and guanine (G), and the pyrimidines cytosine (C) and thymine (T).
• Purines differ from pyrimidines by having a double ring structure (six-membered ring fused to a five-membered ring) while pyrimidines have a single six-membered ring.
• DNA contains deoxyribose sugar while RNA has ribose sugar.
• The carbons of the sugar are numbered 1' to 5'.
• The phosphate group connects to the 5' carbon of the sugar.
• In DNA nucleotides, the 3' carbon of deoxyribose has a hydroxyl (OH) group.
• In RNA nucleotides, the 2' carbon of ribose also has a hydroxyl group.
• The base attaches to the 1' carbon of the sugar.
• Nucleotides link via phosphodiester bonds between the phosphate group on the 5' carbon of one nucleotide and the 3' carbon of the next.
• Polynucleotide chains have a free 5' phosphate at one end and a free 3'-OH at the other, defining the 5' and 3' ends.

Nucleosides vs. Nucleotides

• A nucleoside is a base attached to a five-carbon sugar.
• A nucleotide consists of one to three phosphate groups attached to a nucleoside.

DNA Double Helix

• DNA comprises two strands twisted into a right-handed helix.
• Base pairing occurs between a purine and pyrimidine: A pairs with T, and G pairs with C, as per Chargaff's Rules.
• Adenine and thymine form two hydrogen bonds; cytosine and guanine form three hydrogen bonds.
• The two strands are antiparallel, with the 3' end of one strand facing the 5' end of the other.
• Sugar and phosphate form the backbone, and nitrogenous bases are stacked inside.
• Each base pair is 0.34 nm apart, with each helical turn measuring 3.4 nm, containing 10 base pairs.
• The DNA double helix has a uniform diameter of 2 nm.
• Twisting forms major and minor grooves.

DNA Sequencing

• Dideoxynucleotides (ddNTPs) lack a 3' OH group, terminating chain extension.
• The method uses a predetermined ratio of deoxynucleotides to dideoxynucleotides to create different sized DNA fragments.
• The DNA sample is denatured into two strands.
• The denatured DNA is divided into four tubes with a primer, DNA polymerase, and all four nucleoside triphosphates (A, T, G, and C)
• Limited quantities of one of the four dideoxynucleoside triphosphates (ddCTP, ddATP, ddGTP, and ddTTP) are added to each tube respectively.
• Each of the four dideoxynucleotides carries a different fluorescent label.
• Chain elongation stops when a fluorescent ddNTP is incorporated.
• Electrophoresis separates the fragments, and a laser scanner detects the fluorescent marker to read the sequence.

Gel Electrophoresis

Separates DNA fragments by size using a gel, typically made of agarose.

DNA, negatively charged, moves from the negative to the positive electrode.

Smaller fragments travel farther, separating by molecular weight.

A DNA-specific dye is used to view the separated DNA.

Neanderthal Genome

• Neanderthal DNA was extracted from 40,000-year-old fossil remains.
• Researchers sequenced four billion base pairs.
• The Neanderthal genome showed 2-3% greater similarity to people living outside Africa than to those in Africa.
• Suggests interbreeding occurred between Neanderthals and early modern humans.
• Neanderthals are equally related to people from Papua New Guinea as to those from China or France.
• Some genes, including RUNX2, differ between modern humans and Neanderthals.
• RUNX2 is responsible for differences in cranial structure, rib cage shape, and dental features.
• Evolutionary changes in RUNX2 may have contributed to the origin of modern humans.

Prokaryotic vs. Eukaryotic Genomes

• Prokaryotes contain a single, circular chromosome in the nucleoid region.
• DNA and RNA synthesis occur together in prokaryotic cells.
• E. coli genome size is 4.6 million base pairs.
• DNA is supercoiled to fit inside the bacterial cell, with the help of DNA gyrase.

Eukaryotic Chromosome Packing

• DNA wraps around histones to form nucleosomes, the basic unit of chromatin.
• Histones are evolutionarily conserved proteins rich in basic amino acids, forming an octamer.
• Nucleosomes are linked by linker DNA, creating a "beads on a string" structure.
• Nucleosomes are compacted into a 30-nm fiber with a fifth histone.
• Metaphase chromosomes are highly condensed, approximately 700 nm in width.
• In interphase, heterochromatin is tightly packaged and contains unexpressed genes, while euchromatin is less dense and contains transcribed genes.