Introduction to DNA 2

Screening
1. Immunological Assay
  1. Cells lysed and antigens bound to a matrix
    1. Specific antibodies for the recombinant antigens are then used to identify transformed cells
      1. Primary Antibodies
        These are added first and bind to the recombinant proteins (antigens) from the transformed cells
      2. Secondary Antibodies
        Bind to the already-bound primary antibodies
        Unbound antibodies are washed away before detection
        Labelled to allow detection
        Fluorescently labelled, or to induce a colour change in solution on binding
2. Functional Complementation
  1. To identify the sequence of interest in the library, cells unable to produce the resulting desired product or function are cloned
    1. DNA fragments from the library are taken up into these cells, transforming them
      1. The desired DNA sequence allows the cell to produce the desired product, while the others still can't
        Selecting for the restored function
  2. Example
    1. Mutant cells can be used that do not grow in a certain medium
      1. Recombinant DNA could confer a survival trait, meaning the only ones left are recombinant
        This method can be used in transgenic mice to identify a disease gene
Cloning
1. PCR
  1. Useful when there is not enough material to start with
  2. Allows massive amplification of specific DNA sequences by temperature cycling
  3. DNA is denatured in hot temperatures
    1. Primers are annealed to either side of the region to be amplified
      1. Temperature is cooled to allow the DNA to renature, and Taq polymerase carries out DNA replication to produce an identical stand
        Process is repeated 30-40 times to give millions of copies of the sequence
  4. After two cycles, synthesis only occurs between primers so the new strands are the unit lengths
    1. After three cycles, the new copies are double stranded and the length of the amplicon is the unit length
      1. The unit length copies increase in number with every cycle
        By the end, nearly all the copies are double stranded DNA and unit length products, ready to be used in cloning
Single Nucleotide Polymorphisms
1. Single base pair mutations of normal DNA
2. Fairly frequent and important in inbred populations like domestic animals because they are used for selection purposes
3. Correlated to quantitative trait loci (QTLs) so these can be produce a more desirable phenotypic response after a mutation
4. Can be identified by restriction fragment length polymorphisms (RFLPs)
  1. The base pair change causes the new DNA to become sensitive to a certain restriction endonuclease
    1. Causes different length fragments - an obvious indicator that an SNP has occurred and can be selected for
      1. e.g. a mutation in the myostatin gene causes muscle hypertrophy in Texel sheep. Guanine changes to Adenine, leading to myostatin downregulation and muscle hypertrophy (very muscly sheep)
  2. Method is also very useful in DNA profiling - blood on a suspect's shirt can be analysed to determine whose it is
Gene Transfer
1. Transfer into animal cells is used to study gene function, produce recombinant proteins and to manipulate endogenous gene expression
2. Methods of delivery
  1. Transduction
    1. Virus particles infect mammalian cells and integrate themselves into the genome
  2. Bactofection
    1. Target DNA is carried inside a bacterium, which is taken up by the cell
      1. Digestion releases the DNA
  3. Chemical transfection
    1. DNA and calcium phosphate is taken up by the cell via endocytosis
      1. Usually involved in heat shock
  4. Physical transfection
    1. Microinjection, electroporation and ultrasound
      1. Electroporation increased the permeability by an electric field punching large holes in the membrane. It is very efficient and useful in large plasmids
Gene Targeting
1. Transgenic mice carrying gene mutations are useful for studying human diseases such as cystic fibrosis and cancer
2. Form of in vivo mutagenesis
  1. Where the sequence of a target gene is modified within the cell
  2. Homologous recombination
    1. Two types of vector
      1. Insertion vectors
        Simply insert a gene
      2. Replacement vectors
        Replace sections of DNA
    2. Allows for the generation of animals with human genes
  3. Site-specific recombination
    1. Based on a recombinase enzyme and a stretch of DNA that is recognised by the enzyme
    2. Cre-LoxP system
      1. Cre is an enzyme that causes recombination, and loxP is the site of action
        Swaps the target sequences on two DNA strands - homologous recombination
        Both are derived from bacteriophage P1
        Cre recombinase is an enzyme which catalyses recombination between two loxP sites
        Placing these loxP sites appropriately gives control over recombination, allowing transgenes to be inserted
        Can be modified to only work in certain types of cell
Transgenesis
1. Transgenic mice can be generated by retrieving embryonic stem cells and inserting a transgene by transfection
  1. They are then reinserted into the embryo, which is transferred into a host mouse
    1. After birth, some offspring will show phenotypic signs of the transgene
      1. These are selected and bred again, eventually leading to a homologous +/+ mouse for the transgene
    2. Chimeras are animals with cells contributed from two or more embryos
2. Knockout animals are used to study gene function on specific tissues
  1. Help to understand disease and its resistance and also improve suitability for xenotransplantation
3. Protein Expression Systems
  1. Cell free
    1. Wheat embryos, E.coli lysates
  2. Bacterial
    1. Plasmids and phages
  3. Yeast
    1. Expression vectors (plasmids and yeast artificial chromosomes YACs)
  4. Insect cells
    1. Baculovirus and plasmids
  5. Mammalian
    1. Viral expression vectors (like adenovirus and retrovirus), and stable cell lines (like CHO and HEK293)
Fusion Proteins
1. Created by the joining of two or more genes which originally coded for separate proteins
  1. This is able to carry out functions of both the previous separate proteins
    1. Useful in drug design, creating a pure product that performs many functions
DNA Sequencing - Sanger Method
1. Requires 4 separate reactions - one for each of the four dideoxynucleotides
  1. Each reaction contains a strand of DNA, a labelled primer, DNA polymerase, normal deoxynucleotides and one of the dideoxynucleotides
    1. e.g. ddATP is added to the mix and DNA polymerase starts synthesising a new strand, until ddATP is randomly added to the mix, which stops the reaction due to ddATP not having the 3'-OH group for a phosphodiester bond
      1. This causes, at some point, ddATP to be added to every single point where there is an adenine base at some point in the replication cycle
        Creates lots of different sized fragments, all ending in ddATP
  2. All are radioactively or fluorescently labelled with a different colour
    1. Every possible size fragment has been produced
2. Electrophoresis is carried out separately (but on the same gel) for each dideoxynucleotide reaction, separating the fragments by size order
  1. The order of the sequence can then be read by the labelled nucleotides separated by size order

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