Introduction to DNA

Introduction to DNA

DNA molecule
1. Two long polynucleotide chains
  1. Run antiparallel
  2. Held together by Hydrogen bonds between complementary bases on opposing strands
2. Each strand made of nucleotides
  1. 5 carbon sugar
    1. Ribose in RNA
    2. Deoxyribose in DNA
  2. Nitrogenous base
    1. Pyrimidine
      1. Cytosine
      2. Thymine
      3. Single-ringed base
    2. Purine
      1. Adenine
      2. Guanine
      3. Two-ringed base
    3. 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
  3. One or more phosphate groups
  4. Covalently joined in a sugar-phosphate backbone
    1. 5' phosphate group to 3' OH group on sugar
    2. Phosphodiester bonds
    3. Nucleotides added to the 3' end on lengthening
      1. Molecule of pyrophosphate is released, which is cleaved to pull the reaction forward
  5. Wind around each other in double helix, with a complete turn every 10 base pairs
3. Double helix
  1. Joined together by H bonds in the base (centre), with sugar-phosphate backbone around the outside
Prokaryotic genes
1. Transcription
  1. Starts at a promoter
  2. Ends at a terminator
  3. No further processing of mRNA transcripts
    1. They are translated by the ribosomes as transcription prgresses
Eukaryotic genes
1. Transcription
  1. Starts at a promoter
  2. Ends at a terminator
  3. Primary mRNA transcript is processed, splicing out introns and adding a 5' cap and a 3' polyA tail
    1. This is then translated by the ribosomes into a protein
    2. Different introns can be spliced out, and some can be left, creating multiple RNA transcripts from a single section of DNA
      1. This means that the same gene can code for multiple proteins
Recombinant DNA technology
1. Common cloning strategy
  1. Target DNA from a donor organism is extracted, cut with a restriction endonuclease and ligated using a cloning vector and compatible restriction endonucleases
    1. Recombinant constructs are transferred to a host cell, where cells which do not take up the construct are eliminated using selection protocol
  2. Restriction endonucleases
    1. Cut DNA in two ways
      1. Sticky ends cuts the strands slightly differently, leaving overhangs
      2. Blunt ends leaves strands cut at the same position
    2. Many different enzymes, each of which cuts DNA at a different recognition site, and leaving a different type of cut end
2. Annealing
  1. Sticky ends have a tendency to rejoin (anneal)
  2. Target gene is cut with the same restriction endonuclease as the cloning vector, giving the same sticky ends
    1. If mixed, target DNA can anneal itself into the cloning vector
      1. Vectors can still seal shut without the new DNA, creating a mixture of vectors
        Some with the new DNA, some without
  3. DNA ligase promotes annealing
3. Vectors
  1. Cloning vectors are usually plasmids (circles of DNA) or phages
    1. Plasmids are useful as they have an origin of replication, selectable markers to identify uptake of the new DNA and useful cloning sites
      1. 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
  2. Vectors are usually purified and cut open with restriction endonucleases
DNA Library
1. Collection of portions of a larger genetic element or genome
  1. Created by partially digesting genomic DNA with restriction endonucleases, and then by cloning the fragments into vectors, such as plasmids or phages
  2. To create the fragments, the cell is lysed, and the DNA extracted
    1. DNA then undergoes a partial random cleavage by restriction endonucleases, to create more than one series of identical fragments
      1. Different sizes can be identified and retrieved after different times
    2. To clone the fragments, homogenous replicons are ligated to the ends, containing origins of replication
      1. These are injected into vectors, which are taken up by host cells. Some of which are transformed
  3. To identify a target sequence of interest, a cell is lysed, its DNA extracted and probes attached, which are complementary to the target sequence
    1. These can be DNA or RNA, and are usually over 100bp
    2. A fluorescently labelled marker is also inserted
      1. Can be detected once the marker has bound to the target sequence

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	Criado por Lydia Buckmaster quase 11 anos atrás