Lecture 2_DNA Structure

Lecture 2_DNA Structure - Lecture 2 DNA Structure and...

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Lecture 2: DNA Structure and Manipulation DNA Markers . A genetic marker is a difference in DNA that can be followed from generation to generation. These markers may be phenotypes that can be seen with naked eye or they may changes that are only detectable by methods for analyzing DNA sequence. Chromosomes are composed of DNA ( Fig. 2.1 ). Genes are located in the DNA molecules. In addition to genes there are DNA markers that can be detected by chemical methods such as cutting with restriction nucleases, hybridization with nucleic acid probes and DNA sequencing. DNA cut into discrete fragments by a restriction nuclease can be cloned and amplified in bacterial cells. DNA Structure . DNA is a linear unbranched polymer composed of nucleotide monomers joined by 3’->5’ phosphodiester bonds (see Figs. 2.2 – 2.7 and Table 2.1 ). Each monomer has 3 parts, a 5-carbon sugar (ribose for RNA and 2’-deoxyribose for DNA), a phosphate residue and a nitrogenous base attached to the 1’-position of the sugar. The nitrogenous bases (A, C, G, T/U) are either a pyrimidine (one 6-member ring with 4 carbons and 2 nitrogens) or purine (2 ring structure with 5 carbons and 4 nitrogens; Fig. 2.2 ). Attached to the ring structure are exocyclic groups- amino, keto or methyl. Each position in the ring structure of the bases is numbered- 1,2,3, etc. while the 5 positions in the sugar are numbered 1’, 2’, 3’, etc. The N in the 1-position of a pyrimidine or the N in the 9-position of a purine is attached to the 1’-position of the sugar ( Figs. 2.2, 2.3) A base+ sugar is called a nucleoside (or deoxynucleoside , if the sugar is deoxyribose). Addition of one or more phosphates to a nucleoside converts it to a nucleotide (Table 2.1) . So, for example dA is deoxyadenosine (a deoxynucleoside) and dAMP is deoxyadenosine monophosphate (a nucleotide). In DNA of different organisms, very different amounts of the 4 bases are found ( Table 2.2). However, in double-stranded DNA %A is equal to %T and %G is equal to %C (also called Chargaff’s rule). This is due to Watson-Crick base-pairing - A:T and G:C ( Figs 2.5, 2.6) discussed in Ch. 1. The bases are capable of forming other kinds of base pairs, such as wobble pair and Hoogsteen pairs , which are not present in normal DNA. Two kinds of noncovalent forces hold the two chains together and give it the characteristic right-handed double-helical spiral structure (the usual structure is called “ B-form” of DNA ). There are other shapes possible including a left-handed spiral called the Z-DNA. The twisting of the two strands around each other is uneven resulting in alternative grooves of 2 sizes- major and minor grooves (Fig. 2.5B). The base pairs are on the inside of the helix and the repeating sugar-phosphate backbone on the outside ( Fig. 2.6B ). Each strand has a polarity- 5’phosphate and 3’-OH. The strands in double-helical DNA have opposite polarity ( antiparallel; Fig. 2.7 ). If the sugar-phosphate backbone were fully extended, the base pairs would be about 6.8A apart. However, the bases are quite hydrophobic compounds. They stack up on each other ( Base-stacking ) to keep water out from the inside of the helix. This reduces
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