Nucleic Acids: DNA and RNA

• Nucleic acids are biopolymers essential for all known forms of life.
• They include DNA and RNA.
• Nucleic acids are composed of nucleotide monomers.

Nucleic Acid Fundamentals

• Nucleotides consist of a nitrogenous base, a pentose sugar, and one or more phosphate groups.
• The nitrogenous bases are adenine (A), guanine (G), cytosine (C), thymine (T) in DNA, and uracil (U) in RNA.
• A and G are purines; C, T, and U are pyrimidines.
• The pentose sugar is deoxyribose in DNA and ribose in RNA.
• Nucleotides are linked together by phosphodiester bonds, forming a sugar-phosphate backbone.
• DNA is typically double-stranded, with the two strands held together by hydrogen bonds between complementary bases (A with T, and G with C).
• RNA is typically single-stranded, although it can fold into complex structures through intramolecular base pairing.
• DNA stores genetic information, while RNA plays various roles in gene expression, including mRNA (messenger RNA), tRNA (transfer RNA), and rRNA (ribosomal RNA).

Nucleic Acid Extraction

• Nucleic acid extraction is the process of isolating DNA or RNA from biological samples.
• The goal is to obtain nucleic acids in a pure and intact form, suitable for downstream applications.
• Common extraction methods involve cell lysis, removal of proteins and lipids, and precipitation or binding of nucleic acids.
• Cell lysis can be achieved through mechanical disruption, chemical treatments, or enzymatic digestion.
• Proteins and lipids are typically removed using organic solvents (e.g., phenol-chloroform) or enzymatic digestion (e.g., proteinase K).
• Nucleic acids can be precipitated using ethanol or isopropanol in the presence of salt.
• Alternatively, nucleic acids can be selectively bound to a solid support (e.g., silica membrane or magnetic beads) and then eluted.
• Different extraction methods are optimized for different sample types and downstream applications.

Nucleic Acid Purification

• Nucleic acid purification is the process of removing contaminants from extracted DNA or RNA samples.
• Contaminants can include proteins, lipids, salts, and other cellular components.
• Purification methods include enzymatic treatments (e.g., DNase or RNase digestion), chromatography, and selective precipitation.
• Chromatography involves separating nucleic acids based on size, charge, or affinity using a solid support.
• Commercially available kits often combine extraction and purification steps into a single procedure.
• The purity of nucleic acid samples can be assessed using spectrophotometry, measuring the absorbance ratios at 260/280 nm and 260/230 nm.

Nucleic Acid Sequencing Techniques

• Nucleic acid sequencing is the process of determining the precise order of nucleotides within a DNA or RNA molecule.
• Sanger sequencing was the first widely adopted method, based on chain termination using dideoxynucleotides.
• Next-generation sequencing (NGS) technologies have revolutionized genomics research by enabling massively parallel sequencing.
• NGS platforms include Illumina, Ion Torrent, and PacBio.
• Illumina sequencing uses sequencing by synthesis with fluorescently labeled nucleotides.
• Ion Torrent sequencing detects changes in pH caused by the incorporation of nucleotides.

DNA Sequencing

• DNA sequencing is used to determine the order of nucleotide bases in a DNA molecule.
• Sanger sequencing, also known as chain-termination sequencing, was a widely used method but has been largely replaced by NGS for high-throughput applications.
• NGS methods for DNA sequencing include whole-genome sequencing (WGS), targeted sequencing, and exome sequencing.
• WGS involves sequencing the entire genome of an organism.
• Targeted sequencing focuses on specific regions of interest, such as genes or exons.
• Exome sequencing targets the protein-coding regions of the genome.
• DNA sequencing is used in a wide range of applications, including variant discovery, disease diagnostics, and personalized medicine.

RNA Sequencing

• RNA sequencing (RNA-Seq) is used to study the transcriptome, the complete set of RNA transcripts in a cell or tissue.
• RNA-Seq provides information about gene expression levels, alternative splicing, and novel transcripts.
• RNA-Seq typically involves converting RNA into cDNA (complementary DNA) before sequencing.
• Different RNA-Seq protocols exist, including mRNA-Seq, small RNA-Seq, and total RNA-Seq.
• mRNA-Seq focuses on messenger RNA, which encodes proteins.
• Small RNA-Seq targets small non-coding RNAs, such as microRNAs.
• Total RNA-Seq sequences all RNA molecules in a sample, including ribosomal RNA.
• RNA-Seq data analysis involves mapping reads to a reference genome or transcriptome, quantifying gene expression levels, and identifying differentially expressed genes.

Modified Forms of Nucleic Acids

• Nucleic acids can be modified in various ways, affecting their structure and function.
• DNA modifications include methylation, hydroxymethylation, and glycosylation.
• RNA modifications include methylation, acetylation, and pseudouridylation.
• DNA methylation is the addition of a methyl group to a cytosine base, often associated with gene silencing.
• RNA methylation, such as m6A (N6-methyladenosine), plays a role in RNA processing, stability, and translation.
• Sequencing methods have been developed to detect modified bases, such as bisulfite sequencing for DNA methylation and antibody-based methods for RNA modifications.

Long-Read Sequencing

• Long-read sequencing technologies generate reads that are thousands of base pairs long, compared to the shorter reads produced by Illumina sequencing.
• Long-read sequencing platforms include Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT).
• PacBio sequencing uses single-molecule real-time (SMRT) technology, detecting the incorporation of fluorescently labeled nucleotides.
• ONT sequencing passes DNA or RNA molecules through a nanopore, measuring changes in electrical current to identify the bases.
• Long-read sequencing can span repetitive regions and structural variations in the genome, which are difficult to resolve with short-read sequencing.
• Long reads are valuable for de novo genome assembly, resolving complex genomic regions, and identifying full-length RNA transcripts.