Detailed Study Notes on Nucleic Acids (week 1)

Nucleic Acids

Overview of Nucleic Acids

Nucleic acids are biological molecules that are fundamental to the functioning of living cells. They are polymers composed of monomeric units known as nucleotides.

Structure of Nucleotides

Nucleotides, the building blocks of nucleic acids, have three main components:

  • Sugar molecule: This can be either deoxyribose (in DNA) or ribose (in RNA).

  • Phosphate molecule: The phosphate group is key in linking nucleotides together.

  • Nitrogen-containing molecule: The nitrogenous bases can be adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U). Here, thymine is exclusive to DNA while uracil is found only in RNA. Thus, DNA refers to deoxyribonucleic acid and RNA to ribonucleic acid.

Types of Nucleotides

  • Nucleoside monophosphates: These can undergo further phosphorylation, forming adenosine diphosphate (ADP) and adenosine triphosphate (ATP), which are critical in energy storage and transmission.

The Central Dogma of Molecular Biology

This framework summarizes the flow of genetic information in biological systems, typically represented in a flow chart:

  1. DNA (Gene): The genetic material, undergoes transcription to produce RNA.

  2. Transcription: The process of copying a gene's DNA sequence to make RNA.

  3. RNA: Serves as a template for translation.

  4. Translation: The synthesis of a polypeptide (protein) from mRNA.

  5. Replication: The process of duplicating DNA to pass onto daughter cells.

  6. Reverse transcription: The process where RNA is reverse transcribed back into DNA (not typical in the central dogma).

Nucleotide Polymers and Their Bonding

Nucleotides are linked together by phosphodiester bonds, connecting the 3' carbon atom of one sugar molecule to the 5' carbon atom of another sugar.

Structure of DNA

  • DNA molecules consist of two nucleotide chains that are coiled into a double helix.

  • The strands are held together by base pairs.

  • Base pairing rules: A (adenine) pairs with T (thymine), and G (guanine) pairs with C (cytosine).

DNA Strand Polarity

The two strands of DNA are antiparallel, meaning one strand runs in the 5' to 3' direction while the other runs 3' to 5'. For example:

  • 5’ - GGCTAATCCGTTCGCCCCG - 3’ (sequence of one strand)

  • The complementary sequence can be deduced based on the base pairing rules.

Levels of DNA Packaging

Chromosomes are composed of a single long DNA strand that is complexed with positively charged histone proteins, which help package DNA into a structure known as chromatin. This interaction is essential due to the negative charge of the DNA backbone due to phosphate groups, forming a stable structure necessary for chromosome functionality.

Telomeres

Telomeres are repetitive nucleotide sequences at the ends of linear DNA molecules, serving crucial functions:

  • Protecting chromosome ends from degradation by nucleases.

  • Preventing fusion between other chromosomes.

  • Facilitating complete replication of the DNA ends, ensuring each cell division doesn't shorten important coding regions.

Types of RNA Molecules

There are several types of RNA, each with unique functions:

  • Messenger RNA (mRNA): Carries genetic information from DNA to ribosomes for protein synthesis.

  • Transfer RNA (tRNA): Serves as an adaptor between amino acids and mRNA codons during translation.

  • Ribosomal RNA (rRNA): Forms the core structural and catalytic components of ribosomes.

  • Small nuclear RNAs (snRNAs): Involved in spliceosome function, playing roles in the processing of pre-mRNA.

  • Micro RNAs (miRNAs): Short, single-stranded RNAs that regulate gene expression by blocking complementary mRNAs.

Nucleic Acid Isolation Techniques

Key methods for isolating nucleic acids include:

  • Guanidinium thiocyanate: Used to lyse cells and inhibit RNases during nucleic acid extraction.

  • Phenol-chloroform extraction: Separates DNA from proteins, where DNA partitions to the aqueous phase, and denatured proteins partition to the organic phase.

  • Silica-based purification: Involves selective binding of nucleic acids to silica materials, which offers speed and convenience and is amenable to automation.

  • Alcohol precipitation: Sodium ions and alcohol are used to precipitate DNA by neutralizing its negative charge and facilitating its aggregation.

Quantification of Nucleic Acids

  • Nucleic acids can be quantified by UV absorbance, particularly at 260 nm, where an A260 of 1.0 is equivalent to 50 µg/ml of pure double-stranded DNA. The purity can be assessed using the A260/A280 ratio, ideally around 1.8 to 2.0.

Gel Electrophoresis

This method separates nucleic acid fragments based on size, allowing for visualization and analysis:

  • Agarose gel electrophoresis is commonly used for DNA separation, while polyacrylamide gel electrophoresis allows for finer resolution of smaller fragments.

  • DNA's negative charge causes it to migrate towards the positive electrode under an electric field, with smaller fragments moving faster through the gel matrix than larger ones.