DNA StructureThe 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)

Purine (A+G)

Pyrimidine (T+C)

Bases

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

a nucleotide, a building block of DNA

Note numbering system for carbons in ring

Also note the difference between a ribose used for building DNA and one used for RNA

DNA and RNA chains are made by connecting nucleotides together via chemical bonds

What is this chain RNA or DNA?

• 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

• 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

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

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)

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

1. Lysis of cells to release contents including DNA2. Treatment with EDTA to bind divalent cations3. Proteinase K treatment to digest proteins and tissue away from

DNA4. Separation of DNA from other contaminants in cellular soup using

chemical and physical differences e.g. differential solubilities, precipitation, binding to columns and centrifugation

Cloning a Restriction Fragment into a Plasmid

                                                                                                                                                                                                               

Restriction endonucleases molecular scissors – they cut DNArestriction enzymes are highly specific. They cut DNA only within very precise recognition sequences.

Pst 1 EcoR1 Sma1

Examples of Restriction Enzymes

Enzyme

Organism from which derived

Target sequence(cut at *)5' -->3'

Ava I Anabaena variabilis C* C/T C G A/G G

Bam HI Bacillus amyloliquefaciens G* G A T C C

Bgl II Bacillus globigii A* G A T C T

Eco RI Escherichia coli RY 13 G* A A T T C

Eco RII Escherichia coli R245 * C C A/T G G

Hae III Haemophilus aegyptius G G * C C

Hha I Haemophilus haemolyticus G C G * C

Hind III Haemophilus inflenzae Rd A* A G C T T

Hpa I Haemophilus parainflenzae G T T * A A C

Kpn I Klebsiella pneumoniae G G T A C * C

Sma I Serratia marcescens C C C * G G G

Sal I Streptomyces albus G G * T C G A C

Xma I Xanthamonas malvacearum C * C C G G G

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