DNA as Genetic Material and Central Dogma

• Genes are typically made of DNA and function as units of inheritance
• DNA is considered an information molecule that defines genes in a physical way.

Discovery of DNA as Genetic Material

• Frederick Griffith discovered that Streptococcus pneumoniae could transform from a non-disease-causing strain (R) to a virulent strain (S) in 1923.
• Mixing heat-killed S bacteria with live R bacteria resulted in the R bacteria becoming virulent and able to pass on this trait genetically.
• Avery, MacLeod, and McCarty isolated the "transforming principle" and determined that DNA, not proteins, lipids, or carbohydrates, was responsible for the transformation in 1945.
• Hershey and Chase used bacteriophages with radioactively labeled proteins and DNA, proving that DNA confers heritability.
• Genes are made of RNA in rare cases of some viruses.

Central Dogma of Molecular Biology

• Watson and Crick determined DNA’s structure as a double helix in 1953.
• A DNA double helix consists of two single-stranded DNA (ssDNA) molecules, each a strand composed of nucleotides: guanine (G), adenine (A), thymine (T), and cytosine (C).
• Each strand has a 5' and a 3' end and runs antiparallel to each other, held by hydrogen bonds between the bases.
• G always pairs with C, and A always pairs with T; this base pairing is key to DNA's ability to store and propagate information.

DNA Replication

• DNA replicates through semiconservative replication, using enzymes like DNA helicase and DNA polymerase.
• DNA helicase separates the strands, and DNA polymerase uses each strand as a template to synthesize a new strand following base-pairing rules.
• Semiconservative replication results in new dsDNA molecules composed of one old and one new ssDNA strand.

RNA Transcription

• RNA polymerase uses complementary base-pairing to transcribe information from DNA genes onto RNA molecules.
• RNA is less stable than DNA and acts as a temporary copy of DNA information, using uracil (U) instead of thymine (T).
• RNAs also have a 5' and 3' end and are transcribed in a 5' to 3' direction.
• During transcription, one DNA strand acts as the template strand, while the other is the coding strand.
• The newly transcribed RNA has the same sequence as the coding strand, with U substituted for T.

Protein Translation

• Messenger RNA (mRNA) molecules code for proteins.
• In eukaryotes, mRNAs are transcribed in the nucleus but translated into proteins in the cytoplasm.
• Translation begins at the start codon AUG on the mRNA, after which each set of three bases (codon) codes for an amino acid.
• Amino acids are joined together via peptide bonds to form a polypeptide chain, until a stop codon (UAA, UAG, or UGA) is reached.
• Ribosomes read mRNA in a 5' to 3' direction during translation.

Gene Products Function

• Gene products are typically proteins produced through transcription and translation, which have diverse functions.
• Proteins can act as enzymes, structural components, transport facilitators, hormones, and more.
• Some RNAs, like rRNAs and tRNAs, directly perform biochemical functions.
• Gene expression occurs when a gene is transcribed and translated into a functional product.

DNA and Genes at Scale

• The haploid human genome is approximately 3×10⁹ base pairs (bp) in size, with each chromosome averaging 1.3×10⁸ bp.
• Humans have about 20,000 genes, with each chromosome containing roughly 1,000 genes.
• Genes typically range from 10³ to 10⁴ base pairs, but some can be much larger, such as the human dystrophin gene at 2 x 10⁶ bp.
• Duchenne muscular dystrophy (DMD) results from mutations in dystrophin and is a sex-linked trait on the X chromosome, affecting males more.
• Most eukaryotic genes contain introns, which are removed from the RNA transcript via splicing; intergenic regions are non-coding sequences between genes.

Rethinking Familiar Terms

• Alleles are different versions of the same gene with variations in their DNA sequences that may or may not alter the amino acid sequence of the encoded protein.
• Mutations are altered versions of a gene that result in a markedly different phenotype.
• Variations or variants is another term used instead of mutation.

Drosophila example

• A mutant allele of the shibire gene codes for a heat-sensitive protein required for synaptic transmission.
• Flies with the shibire mutation become paralyzed when warmed up due to defective synaptic signaling.

Definition of a Physical Gene

• The physical material of genes is DNA, with RNA as an intermediate, and protein as the gene product that provides function.
• A typical mammalian genome is much larger than the portion accounted for by genes.
• Most of the genome does not contain genes based on the estimate of 20,000 genes at 10,000 bp each, compared to the 3×10⁹ bp genome size.

Bacterial Gene Structure

• Bacterial genes are useful for study and as tools, sharing principles with eukaryotic genes but also showing key differences.
• The bacterial gene includes: a promoter to target RNA polymerase, a transcription terminator, a Shine-Dalgarno sequence for ribosome loading, a start codon (AUG), a coding sequence, and a translation stop codon.

Identifying Genes Based on DNA Sequences

• Genes are classically identified by their function and the phenotypic changes caused by mutations.
• Genes of unknown function are now detected using DNA sequence patterns.
• Open reading frames (ORFs), stretches lacking stop codons, indicate potential genes in bacteria, where random sequences should have a stop codon every 21 codons. The probability of a random ORF of 100 codons is low
• Eukaryotic gene identification is more complex due to non-coding intergenic regions and introns.
• Introns break open reading frames, making it harder to distinguish genes from non-coding sequences.
• Introns are removed and exons are joined during splicing to form mature mRNA.

Eukaryotic DNA

• Yeast genes contain few introns and are closely packed, unlike Drosophila and human genes, which are spread apart and interrupted by many introns.

Identification of Eukaryotic Genes

• Examining mRNA can identify eukaryotic genes.
• Reverse transcriptase converts mRNAs into complementary DNAs (cDNAs).
• Sequencing cDNAs and comparing them to genomic DNA identifies and locates genes.
• Expressed sequence tags (ESTs) are randomly sequenced cDNAs that suggest gene locations.
• RNAseq is a modern technology faster than cDNA approaches for gene identification.

DNA Sequencing Background

• DNA sequencing determines the order of nucleotides in a DNA fragment.
• DNA is double-stranded with directional strands, having a 5' and 3' end.
• Strands run anti-parallel and are held by hydrogen bonds.
• DNA polymerase synthesizes a copy of a DNA template using dATP, dGTP, dCTP, and dTTP (dNTPs).
• DNA polymerase requires dNTPs, a template strand, and a primer with a free 3' hydroxyl group.
• New DNA is synthesized by elongating an existing strand at its 3' end, progressing in a 5' to 3' direction relative to the new strand.

DNA Sequencing Details

• Sanger sequencing, invented by Frederick Sanger in 1977, is a DNA sequencing technology.
• dsDNA strands are separated by heating, and a short primer anneals to the ssDNA.
• DNA polymerase, dNTPs, and a small amount of dideoxyribonucleotide triphosphates (ddNTPs) are added.
• ddNTPs, each with a unique fluorophore, lack a 3' OH group and act as chain terminators.
• Reactions are initiated, and samples are denatured and separated using a high-resolution gel system.
• The DNA sequence is read from the gel by scanning it with a laser and visualizing fluorescent light.
• Elongation stops with ddNTP incorporation, creating labeled ssDNA strands that terminate at different positions.
• Electrophoresis separates fragments by size on a gel, and a computer reads the colored bands to determine the sequence.

Polymerase Chain Reaction (PCR)

• A large amount of DNA is needed for Sanger sequencing.
• PCR, developed by Kary Mullis, is an in vitro method used to obtain DNA. This avoids molecular cloning.
• Steps include extracting DNA, adding primers, melting and cooling DNA for primer annealing, and adding DNA polymerase and nucleotides.
• Taq polymerase, a heat-stable enzyme, is used to withstand heating cycles.
• Each PCR cycle doubles the amount of the DNA segment to give exponential increase.
• After 10 cycles there is approximately a 1000-fold increase and after 20 cycles approximately a 10^6-fold increase.
• Resulting PCR product can be used for DNA sequencing.
• Starting with a single molecule of dsDNA, 38 cycles of PCR would be needed to obtain 1 μg of a 1 kb long DNA
• Each cycle of PCR takes approximately 1-2 minutes

NGS Technologies

• NGS technologies have made DNA sequencing faster and cheaper compared to Sanger sequencing.
• Illumina sequencing involves fragmenting an entire genome into millions of small pieces and affixing them to a flow cell.
• Fragments are amplified in situ by solid-phase bridge PCR, forming "colonies" of DNA clones.
• Labeled dNTPs with a removable blocking group are used instead of ddNTPs, and a camera takes pictures of the flow cell.
• Blocking groups and fluorophores are removed, and new sequencing reagents are added.
• A computer assembles the sequences from individual fragments into a complete sequence.
• The process is slower on a strand-by-strand basis but faster overall due to parallel processing of millions of fragments.
• Sequencing depth (number of times a DNA sequence exists in different fragments that are read) can overcome the error rate.

Illumina Sequencing Steps

• During cluster generation, steps (1)-(3) are known as bridging PCR.
• Each cluster contains identical copies (or clones) of ssDNA that are affixed to a specific physical location on the surface of the flow cell.
• Sequencing by synthesis in Illumina sequencing depends on a 3 nucleotide sequence 5'-CGG-3'; in reality it will be much longer (~20-30 nt).
• Technologies such as Illumina sequencing are now the preferred method for most types of large-scale DNA sequencing.

Other NGS Technologies

• RNA sequencing (RNAseq) can determine how much a particular gene is expressed via transcription and determine the number of reads of a particular RNA sequence.
• Chromatin immunoprecipitation sequencing, or ChIPseq is useful for finding out what DNA sequences protein X binds to.
• Nanopore sequencing does not use DNA polymerase and can be used to replace Sanger sequencing in some routine applications.
• Nanopore sequencing has the advantage of being able to detect DNA that has been chemically modified, such as methylated bases.