Recombinant DNA Technology

Recombinant DNA Technology

1. Producing DNA Fragments

   • Many techniques developed to isolate, clone and transfer genes into an organism.
   • Recombinant DNA = DNA of 2 different organisms
   • Transgenic/GMO = organisms with recombinant DNA
   • Human diseases that result from a lack of a protein (faulty genes) used to be treated by injecting the protein into the body
     • new technique of rcombinnant DNA avoids rejection / infection
     • Genetic code is universal so DNA is accepted and translated as normal
   • Process of making a gene
     • Using reverse transcriptase
       • Retroviruses use RNA so are able to synthesis DNA from RNA using reverse transcriptase
       • 1) Cells identified containing mRNA
       • 2) mRNA coding for protein seperated
       • 3) mRNA acts as a template on which a complementary strand of cDNA is made
       • 4) single stranded cDNA is isolated by hydrolysis
       • 5) Double stranded DNA is formed by the template using DNA polymerase
     • Using Restriction Enzymes
       • some bacteria protect themselves by producing enzymes that cut up viral DNA = restriction enzymes
       • restriction enzymes cut the DNA at specific region --> recognition sites
         • Blunt ends
         • Sticky ends
           • leaving exposed bases
         • sites are always a palindrome (read the sites both ways)
     • Gene Machine
       • 1) The desired nucleotide sequence is determined from the protein we want.
         • 2) Amino acid sequence determined and so mRNA is determined and cDNA worked out
           • 3) Desired nucleotide base is fed onto a computer
       • 4) Sequence is checked for biosafety + biosecurity to meet ethical and safety standards
         • 5) Each nucleotide sequence is added one at a time in the sequence
           • 6) All joined together to make a gene with no introns
             • 7) PCR constructs complementary strand to make double stranded DNA
               • 8) Using sticky ends the gene is inserted into a plasmid
                 • 9) Genes are checked and errors rejected
         • 5) Computer makes small overlapping sequences called oligonucleotides
       • ADV = any sequence can be produced , Quick, free of introns
     • 1) Isolation of gene from DNA
     • 2) Insertion of gene into vector
     • 3) Transformation into host
     • 4) Identification of the host that have the gene by use of gene markers
     • 5) Growth of the host cells

2. In vivo Gene Cloning

   • After the DNA has been cut into fragments we find which fragment contains the desired gene by using DNA probes
   • In vivo = living things
   • Importance of sticky ends =
     • Restriction enzyme cuts the DNA at a specific recognition site producing sticky ends
       • This is complementary to another strand cut by the same enzyme as the same recognition site
         • DNA ligase joines both sections
   • Preparing the DNA for insertion
     • Need to add a promoter (length of DNA for the RNA polymerase to attach to)
     • Need to add terminator (length of DNA stimulating the end of transcription
     • Vector = carrying unit used to transport DNA into a host
     • DNA will be inserted into a vector
     • Plasmids act as the vector
     • Plasmids contain genes for antibiotic resistance so restriction enzymes are used to break up these plasmids
       • When mixed together, DNA fragments (cut by the same restriction enzyme) bind
         • DNA ligase attaches DNA to plasmid
   • Useful when putting genes into other organisms (vectors)
   • No risk of contamination
   • Very accurate
   • Cuts out specific genes
   • Bacteria can produce large quantities of gene product

3. Introduction of DNA into host cells

   • Process = transformation
   • Plasmid containing DNA is put into bacterial cells
   • Vector + Cells are mixed in a medium with calcium ions and heat
     • Makes the membrane permeable allowing plasmids to pass through
   • Not all bacterial posses the DNA as..
     • Few cells take up the plasmid
     • Some plasmids will have closed up again
     • DNA sometimes joins itself and forms into own plasmid

4. Locating the Gene

   • Uses the gene for antibiotic resistance
   • 1) Bacteria is grown on medium containing ampicillin
   • 2) Bacteria with plasmid will be resistant due to having the antibiotic resistant gene
   • 3) Bacteria without the plasmid will die due to not having the resistant gene
   • Effective however some plasmids will have closed up before incorperating new gene
     • Should use a second gene on plasmid (marker gene)
       • Antibiotic resistance
         • Same method as before but Gene AB-B is cut up in order to incorperate the desired gene
           • Therefore won't be resistant to B but will be resistant to A, as before
             • When grown on culture A, those with plasmid will survive
             • When grown on culture B, those with plasmid will die
       • Fluorescence
         • More rapid method
         • Transfer of fluorescent protein from jellyfish
           • Gene of interest is inserted in middle of fluorescent gene
             • Cells not fluorescent have the gene
       • Enzyme Markers
         • Genes that produces lactase will turn substrate blue
         • Gene of interest inserted into middle of lactase gene, those not blue have the gene

5. In Vitro Gene Cloning

   • Polymerase Chain Reaction
   • Requires...
     • DNA fragments
     • DNA Polymerase
     • Primers
     • Nucleotides
   • 1) Everything is heated to 95 dC to break H bonds and separate strands
   • 2) Cooled to 55 dC causing primers to join ends of DNA (complementary base pairing)
     • Prevents strands attaching back together
   • 3) Increased to 72 dC for optimum for DNA polymerase to add nucleotides to seperate strands
   • Extremely rapid
   • Doesn't require living cells

6. Locating Genes

   • Can treat diseases
     • Only if know exactly where on DNA sequence located
   • DNA Probes
     • Short single stranded DNA with a label
     • Radioactively Labelled
       • Nucleotides with isotopes 32P and identified with X Ray film
     • Fluorescently Labelled
       • emits light under certain conditions
     • 1) Probe is made from bases complementary to gene of interest
       • Have to know the base sequence of gene of interest
     • 2) Double stranded DNA is seperated
     • 3) Single stranded DNA is mixed with the probe, they anneal
       • DNA Hybridisation
     • 4) Site at which probe binds can be identified
   • Locating specific alleles
     • Determining whether someone has a mutant allele causing a disease
     • 1) Determine the sequence of nucleotides of the mutant allele
     • 2) DNA produced that has sequence complementary to mutant allele
     • 3) DNA probe is made by adding a marker
     • 3) PCR forms multiple copies
     • 4) Single stranded DNA of individual is mixed with newly made probe
     • 5) If contains allele, probe will bind
     • 6) DNA washed and tested for detection

7. Genetic Screening

   • Identify carriers which are heterozygous for a recessive allele
     • Potential to pass on disease
   • Detection of oncogenes
     • enables people to make life choices that can decrease the chances of cancer
     • Oncogenes prevent tumour suppressant genes working and initiate uncontrolled cell division
   • Personalised Medicine
     • Based on individuals genotype drugs can be more/less effective
     • Personalised dosage
     • Save money
   • Genetic Counselling
     • Revise family history - likelihood of passing on
     • Advice / information enabling people to make personalised decisions
   • Genetic Fingerprinting
     • Relies on the fact DNA contains non-coding parts
       • Different for everyone yet similar in closely related
     • 1) Extraction of DNA fragment and increase sample by PCR
     • 2) Sample is cut by restriction enzymes
     • 3) Strands are immersed in alkali and separated by gel elctrophoresis
       • Fragments separated according to size
         • Labelled so can see positions
       • Smaller fragments move further towards negative charge
     • 4) Radioactive / Fluorescent Probes are hybridised (specific base pairing)
       • Excess washed off
     • 5) Xrays / fluorescent light is put over the nylon membrane
     • Pattern of bands is unique to everyone
     • Used for ...
       • Paternity Tests
         • A childs DNA bands will match atleast one band in either of the parents fingerprint
         • more closely related the more similar the fingerprint
       • Forensic Science
         • DNA found at the scene can be tested to match the suspects
       • Medical Diagnosis
         • Huntingtons disease results from a large number of base sequence repeats
       • Plant and animal breeding
         • Used in breeding programmes to prevent undesirable breeding
         • Ensure pedigree

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