Introduction to DNA 2

Introduction to DNA 2

1. Screening

   • Immunological Assay
     • Cells lysed and antigens bound to a matrix
       • Specific antibodies for the recombinant antigens are then used to identify transformed cells
         • Primary Antibodies
           • These are added first and bind to the recombinant proteins (antigens) from the transformed cells
         • 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
   • Functional Complementation
     • To identify the sequence of interest in the library, cells unable to produce the resulting desired product or function are cloned
       • DNA fragments from the library are taken up into these cells, transforming them
         • The desired DNA sequence allows the cell to produce the desired product, while the others still can't
           • Selecting for the restored function
     • Example
       • Mutant cells can be used that do not grow in a certain medium
         • 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

2. Cloning

   • PCR
     • Useful when there is not enough material to start with
     • Allows massive amplification of specific DNA sequences by temperature cycling
     • DNA is denatured in hot temperatures
       • Primers are annealed to either side of the region to be amplified
         • 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
     • After two cycles, synthesis only occurs between primers so the new strands are the unit lengths
       • After three cycles, the new copies are double stranded and the length of the amplicon is the unit length
         • 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

3. Single Nucleotide Polymorphisms

   • Single base pair mutations of normal DNA
   • Fairly frequent and important in inbred populations like domestic animals because they are used for selection purposes
   • Correlated to quantitative trait loci (QTLs) so these can be produce a more desirable phenotypic response after a mutation
   • Can be identified by restriction fragment length polymorphisms (RFLPs)
     • The base pair change causes the new DNA to become sensitive to a certain restriction endonuclease
       • Causes different length fragments - an obvious indicator that an SNP has occurred and can be selected for
         • 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)
     • Method is also very useful in DNA profiling - blood on a suspect's shirt can be analysed to determine whose it is

4. Gene Transfer

   • Transfer into animal cells is used to study gene function, produce recombinant proteins and to manipulate endogenous gene expression
   • Methods of delivery
     • Transduction
       • Virus particles infect mammalian cells and integrate themselves into the genome
     • Bactofection
       • Target DNA is carried inside a bacterium, which is taken up by the cell
         • Digestion releases the DNA
     • Chemical transfection
       • DNA and calcium phosphate is taken up by the cell via endocytosis
         • Usually involved in heat shock
     • Physical transfection
       • Microinjection, electroporation and ultrasound
         • Electroporation increased the permeability by an electric field punching large holes in the membrane. It is very efficient and useful in large plasmids

5. Gene Targeting

   • Transgenic mice carrying gene mutations are useful for studying human diseases such as cystic fibrosis and cancer
   • Form of in vivo mutagenesis
     • Where the sequence of a target gene is modified within the cell
     • Homologous recombination
       • Two types of vector
         • Insertion vectors
           • Simply insert a gene
         • Replacement vectors
           • Replace sections of DNA
       • Allows for the generation of animals with human genes
     • Site-specific recombination
       • Based on a recombinase enzyme and a stretch of DNA that is recognised by the enzyme
       • Cre-LoxP system
         • 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

6. Transgenesis

   • Transgenic mice can be generated by retrieving embryonic stem cells and inserting a transgene by transfection
     • They are then reinserted into the embryo, which is transferred into a host mouse
       • After birth, some offspring will show phenotypic signs of the transgene
         • These are selected and bred again, eventually leading to a homologous +/+ mouse for the transgene
       • Chimeras are animals with cells contributed from two or more embryos
   • Knockout animals are used to study gene function on specific tissues
     • Help to understand disease and its resistance and also improve suitability for xenotransplantation
   • Protein Expression Systems
     • Cell free
       • Wheat embryos, E.coli lysates
     • Bacterial
       • Plasmids and phages
     • Yeast
       • Expression vectors (plasmids and yeast artificial chromosomes YACs)
     • Insect cells
       • Baculovirus and plasmids
     • Mammalian
       • Viral expression vectors (like adenovirus and retrovirus), and stable cell lines (like CHO and HEK293)

7. Fusion Proteins

   • Created by the joining of two or more genes which originally coded for separate proteins
     • This is able to carry out functions of both the previous separate proteins
       • Useful in drug design, creating a pure product that performs many functions

8. DNA Sequencing - Sanger Method

   • Requires 4 separate reactions - one for each of the four dideoxynucleotides
     • Each reaction contains a strand of DNA, a labelled primer, DNA polymerase, normal deoxynucleotides and one of the dideoxynucleotides
       • 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
         • 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
     • All are radioactively or fluorescently labelled with a different colour
       • Every possible size fragment has been produced
   • Electrophoresis is carried out separately (but on the same gel) for each dideoxynucleotide reaction, separating the fragments by size order
     • The order of the sequence can then be read by the labelled nucleotides separated by size order