DNA Structure The deoxyribonucleic acid, DNA, is a long chain of nucleotides which consist of

1. DNA Structure The deoxyribonucleic acid, DNA, is a long chain of nucleotides which consist of Deoxyribose (a pentose = sugar with 5 carbons) Phosphoric Acid Organic (nitrogenous) bases (Purines - Adenine and Guanine, or Pyrimidines -Cytosine and Thymine)

2. Bases Purine (A+G) Pyrimidine (T+C)

3. a nucleotide, a building block of DNA. It is a phosphate ester of a nucleoside Nucleotide Nucleoside

4. a nucleotide, a building block of DNA

5. Note numbering system for carbons in ring Also note the difference between a ribose used for building DNA and one used for RNA

6. DNA and RNA chains are made by connecting nucleotides together via chemical bonds What is this chain RNA or DNA?

7. four different types of nucleotide possible in a DNA sequence, adenine, cytosine, guanine and thymine (ATCG) • Nucleotides are situated in adjacent pairs in the double helix. • Thymine and adenine can only make up a base pair • Guanine and cytosine can only make up a base pair

9. Double-stranded DNA is simply two chains of single- stranded DNA, positioned so their "bases" can interact with each other. • The sugar-and-phosphate 'backbone' is red, and the bases are blue. • the two strands travel in opposite directions; "anti-parallel". • The bases in the middle "pair up" with bases on the opposite strand, A+T, G+C • Hydrogen bonds hold stucture together

12. Genome- entire complement of genetic information. • Includes coding and non coding • Genes (exons and introns) • Alleles are different gene forms • Useful DNA for doing genome analysis

14. Plasmid- extra chromosomal DNA, found naturally mainly in bacteria • covalent closed circle, double stranded DNA • Non essential • Replicates independently • Occurs naturally in bacteria, • Molecular biologists recognise use and made them their own • Used as cloning vectors i.e. to transfer DNA between bacteria Recombinant plasmids made by molecular biologists have been designed to carry foreign DNA into bacterial cells. They have • unique restriction enzyme sites. Usually many different ones in a polylinker site • origin of replication for bacteria • selectable marker (often antibiotic resistance)

16. DNA isolation and purification • Important to obtain clean intact DNA in sufficient quantities to work with • Always do on ice Most purification procedures include many of the following steps • Lysis of cells to release contents including DNA • Treatment with EDTA to bind divalent cations • Proteinase K treatment to digest proteins and tissue away from DNA • Separation of DNA from other contaminants in cellular soup using chemical and physical differences e.g. differential solubilities, precipitation, binding to columns and centrifugation

17. Cloning a Restriction Fragment into a Plasmid

18. Restriction endonucleases molecular scissors – they cut DNA restriction enzymes are highly specific. They cut DNA only within very precise recognition sequences. Pst 1 EcoR1 Sma1

20. Examples of Restriction Enzymes

21. DNA ligase • Enzyme • is molecular glue- sticks DNA together • H bonds are not enough to hold sticky ends together. A means of reforming the internucleotide linkage between 3’OH and 5’phosphate groups is required and ligase does this