Molecular Biology

Molecular Biology Nucleic acids Chemical composition and bonds Glycosidic bond This is why they are acidic Ribose vs. deoxyribose • Positively charged ions (Na+ or Mg2+) and peptides with positively charged side chains can associate with DNA • Eukaryotic DNA, for example, is complexed with histones, which are positively charged proteins, in the cell nucleus. Nitrogenous bases Glycosidic bond Nucleotides vs. Nucleosides Nucleotides vs. Nucleosides Nucleic acid polymer • A letter d can be added to indicate a deoxyribonucleotide residue. • for example, dG is substituted for G. • The deoxy analogue of a ribooligonucleotide would be d(GACAT). DNA structure Specific base-pairing A = T; G = C; Pur = pyr Complementary A double helix Backbone vs. side chains Antiparallel Stable Flexible Groovings Stability vs. flexibility Base pairing DNA structure Specific base-pairing A = T; G = C; Pur = pyr Complementary A double helix Backbone vs. side chains Antiparallel Stable Flexible Groovings Stability vs. flexibility DNA coiling The bacterial circular DNA can be coiled by rotating it clockwise (negative supercoiling) or counterclockwise (positive supercoiling). Supercoils can be added or removed by enzymes called topoisomerases: Class I topoisomerases cut the phosphodiester backbone of one strand of DNA. Class II topoisomerases cut both strands of DNA. In bacteria, it is called DNA gyrase. In eukaryotes… In eukaryotes, DNA coiling is utilized for DNA packaging and regulation of gene activity. Eukaryotic DNA is complexed with a number of proteins. Chromatin = DNA molecule + proteins The principal proteins in chromatin are the histones. Nucleosomes The protein core is an octamer (two molecules of histones H2A, H2B, H3, and H4). A linker DNA/spacer region connects the octamer-DNA complexes. A nucleosome consists of DNA wrapped around a histone core. H1 is bound to the the octamer and wrapped DNA (a chromatosome). Histones are positively cahrged facilitating DNA interaction and charge neutralization. Light absorbance of nucleic acids Aromatic pyrimidines and purines can absorb UV light The peak absorbance is at 260 nm wavelength The absorbance of nucleic acids at 260 nm (A260) is constant dsDNA: A260 of 1.0 = 50 ug/ml ssDNA: A260 of 1.0 = 30 ug/ml ssRNA: A260 of 1.0 =40 ug/ml Reason for ss vs. ds absorbance: • Stacked bases, vs. unstacked bases What is the concentration of a double stranded DNA sample diluted at 1:10 and the A260 is 0.1? DNA concentration = 0.1 x 10 x 50 µg/ml = 50 µg /ml hyperchromicity Observation of denaturation The transition temperature, or melting temperature (Tm). Factors influencing Tm G·C pairs Hydrogen bonds Base stacking pH Salt and ion concentration Destabilizing agents (alkaline solutions, formamide, urea) Central dogma of biology Genetic information must be preserved via DNA replication. Information must be translated into action makers (proteins) via transcription and translation. RNA Sequence is dictated by DNA sequence. RNA Consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one pentose and the 5’-OH of the next The pentose unit is β-D-ribose (it is 2-deoxy-D-ribose in DNA) The pyrimidine bases are uracil and cytosine (they are thymine and cytosine in DNA) In general, RNA is single stranded (DNA is double stranded). Types of RNA Hybridization DNA from different sources can form double helix as long as their sequences are compatible (hybrid DNA) 19 Techniques Gel electrophoresis Hybridization techniques 20 Gel electrophoresis The length and purity of DNA molecules can be accurately determined by the gel electrophoresis Direction DNA travels + wells - Detection The DNA molecules of different lengths will run as "bands“ - DNA is stained (that is, colored) with a dye (ethidium bromide) or radioactively labeled (32P) 1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp + Hybridization techniques Hybridization reactions can occur between any two singlestranded nucleic acid chains provided that they have complementary nucleotide sequences Hybridization reactions are used to detect and characterize specific nucleotide sequences 25 Probes A probes is a short sequence of single stranded DNA (an oligonucleotide) that is complementary to a small part of a larger DNA sequence Hybridization reactions use labeled DNA probes to detect larger DNA fragments 26 Southern blotting This technique is a combination of DNA gel electrophoresis and hybridization Used to detect: the presence of a DNA segment complementary to the probe the size of the DNA fragment 27 1 2 1 2 1 2 : probe Electrophoresis Southern blot