Introduction to DNA

Introduction to DNA

1. DNA molecule

   • Two long polynucleotide chains
     • Run antiparallel
     • Held together by Hydrogen bonds between complementary bases on opposing strands
   • Each strand made of nucleotides
     • 5 carbon sugar
       • Ribose in RNA
       • Deoxyribose in DNA
     • Nitrogenous base
       • Pyrimidine
         • Cytosine
         • Thymine
         • Single-ringed base
       • Purine
         • Adenine
         • Guanine
         • Two-ringed base
       • A to T and C to G ensures that each base pair is of a similar size, allowing efficient packing in the centre of the helix
     • One or more phosphate groups
     • Covalently joined in a sugar-phosphate backbone
       • 5' phosphate group to 3' OH group on sugar
       • Phosphodiester bonds
       • Nucleotides added to the 3' end on lengthening
         • Molecule of pyrophosphate is released, which is cleaved to pull the reaction forward
     • Wind around each other in double helix, with a complete turn every 10 base pairs
   • Double helix
     • Joined together by H bonds in the base (centre), with sugar-phosphate backbone around the outside

2. Prokaryotic genes

   • Transcription
     • Starts at a promoter
     • Ends at a terminator
     • No further processing of mRNA transcripts
       • They are translated by the ribosomes as transcription prgresses

3. Eukaryotic genes

   • Transcription
     • Starts at a promoter
     • Ends at a terminator
     • Primary mRNA transcript is processed, splicing out introns and adding a 5' cap and a 3' polyA tail
       • This is then translated by the ribosomes into a protein
       • Different introns can be spliced out, and some can be left, creating multiple RNA transcripts from a single section of DNA
         • This means that the same gene can code for multiple proteins

4. Recombinant DNA technology

   • Common cloning strategy
     • Target DNA from a donor organism is extracted, cut with a restriction endonuclease and ligated using a cloning vector and compatible restriction endonucleases
       • Recombinant constructs are transferred to a host cell, where cells which do not take up the construct are eliminated using selection protocol
     • Restriction endonucleases
       • Cut DNA in two ways
         • Sticky ends cuts the strands slightly differently, leaving overhangs
         • Blunt ends leaves strands cut at the same position
       • Many different enzymes, each of which cuts DNA at a different recognition site, and leaving a different type of cut end
   • Annealing
     • Sticky ends have a tendency to rejoin (anneal)
     • Target gene is cut with the same restriction endonuclease as the cloning vector, giving the same sticky ends
       • If mixed, target DNA can anneal itself into the cloning vector
         • Vectors can still seal shut without the new DNA, creating a mixture of vectors
           • Some with the new DNA, some without
     • DNA ligase promotes annealing
   • Vectors
     • Cloning vectors are usually plasmids (circles of DNA) or phages
       • Plasmids are useful as they have an origin of replication, selectable markers to identify uptake of the new DNA and useful cloning sites
         • e.g. Restriction endonucleases can be used that insert the DNA into a section of the plasmid that codes for resistance to a certain bacteria
           • This way, the cells that have taken up recombinant plasmids lose their resistance and can be detected.
           • Other plasmid vectors are available with LacZ and LacI genes
     • Vectors are usually purified and cut open with restriction endonucleases

5. DNA Library

   • Collection of portions of a larger genetic element or genome
     • Created by partially digesting genomic DNA with restriction endonucleases, and then by cloning the fragments into vectors, such as plasmids or phages
     • To create the fragments, the cell is lysed, and the DNA extracted
       • DNA then undergoes a partial random cleavage by restriction endonucleases, to create more than one series of identical fragments
         • Different sizes can be identified and retrieved after different times
       • To clone the fragments, homogenous replicons are ligated to the ends, containing origins of replication
         • These are injected into vectors, which are taken up by host cells. Some of which are transformed
     • To identify a target sequence of interest, a cell is lysed, its DNA extracted and probes attached, which are complementary to the target sequence
       • These can be DNA or RNA, and are usually over 100bp
       • A fluorescently labelled marker is also inserted
         • Can be detected once the marker has bound to the target sequence