What defines the molecular structures of DNA and RNA?
• The DNA double helix is formed through pairing of nucleotides. Adenine bases hydrogen bond with thymine bases. Guanine bases hydrogen bond with cytosine bases. The two strands of DNA are oriented in an antiparallel fashion.
• Stacking of adjacent base pairs in a helix stabilizes the helix through van der Waals interactions and the hydrophobic effect. Base stacking is the primary source of stabilization for the double helix.
• The melting temperature (Tm) of DNA is influenced by its base composition, the ionic strength of the solution, and the length of the DNA molecule.
• The 2’-hydroxyl group on RNA can lead to an autocleavage reaction. This property makes RNA unsuitable as a storage form for genetic material. DNA, which lacks the 2’-hydroxyl group, is a chemically more stable molecule.
How are genomes organized and how can genomics be used as a forensic tool?
● Histone proteins facilitate the condensation of eukaryotic DNA. Nucleosomes are DNA–histone complexes, which can be further condensed to form chromatin. Chromatin can be found as either euchromatin (less condensed) or heterochromatin (more condensed). The switch between these states is influenced by covalent modifications of the histones or DNA.
● DNA in chromosomes is condensed more than 10,000- fold compared to the extended double-helical structure. A mitotic chromosome consists of a central region of heterochromatin called the centromere. The ends of chromosomes contain telomeres, which are repetitive DNA sequences. Telomeres function to maintain the length of chromosomes after replication.
● Eukaryotic RNA transcripts are usually modified by 5’ capping, 3′ polyadenylation, and splicing and contain both noncoding regions (introns) and coding regions (exons). Alternative splicing of these exons generates multiple mRNA and protein products from a single gene.
● Bioinformatic tools can be used to compare the genomes of different organisms and predict the structure of a gene or the function of a protein. Single nucleotide polymorphisms (SNPs) in DNA can be used to diagnose disease and to explore genetic diversity.
What are some of the most common methods in nucleic acid biochemistry?
● Bacteria contain enzymes that protect against bacteriophage infection: DNA sequence–specific DNA methylases and restriction endonucleases. DNA methylation blocks restriction endonuclease cleavage at recognition sites; thus, bacteriophage DNA is cleaved, but the bacterial DNA is protected.
● The Sanger DNA sequencing method is based on chain termination through the incorporation of dideoxynucleotides, which lack a hydroxyl group at the ribose 3’-position and block phosphodiester bond formation.
● PCR is an automated method of amplifying DNA sequences. Temperature cycling is used to denature and reanneal the DNA, whereas thermostable DNA polymerases extend the DNA primers.
● The complexity and variety of RNA transcripts in a cell sample can be analyzed by gene expression microarrays and by RNA-seq. Gene expression microarrays measure the abundance of predetermined sequences, whereas RNA-seq provides unbiased data on all transcripts.
● The bacterial CRISPR-Cas9 system consists of an RNA–protein complex that recognizes target DNA using a complementary nucleotide RNA sequence of at least 20 nucleotides. Components of the CRISPR-Cas9 system include (1) single-guide RNA (sgRNA), (2) Cas9 endonuclease, and (3) a PAM sequence in the region of the target DNA consisting of three to five nucleotides.