Molecular Tools and Techniques

Molecular Tools and Techniques

1. Restriction Enzymes

   • bacterial enzyme
   • recognise a short sequence of bases in a DNA molecule
     • cuts the DNA at this recognition site
       • 'molecular scissors'
       • blunt ends
         • do not ligate
       • sticky ends
         • staggered cut
           • short, single-stranded ends
         • DNA molecule with sticky ends another DNA molecule with complementary sticky ends
           • two pieces join up
             • reform hydrogen bonds
             • DNA ligase stabilises recombined molecule
               • 'molecular glue'
     • different restriction enzymes recognise different sites
       • cut the DNA into different sized fragments
         • unique banding pattern on a gel
           • gives DNA profile of that individual

2. Gel Electrophoresis

   • DNA is cut into many fragments of different lengths by restriction enzymes
     • fragments are sequenced according to size and charge
   • fragments are placed into the wells of an agarose gel
     • electric field is set up
       • negatively charged DNA fragments are drawn to the positive terminal
     • highly porous, acts like a sieve
       • shortest DNA fragments are able to move more quickly and easily through pores
         • travel the furthest distance
       • more difficult for longer fragments to pass through pores
         • lag behind shorter fragments
     • gel is injected with a stain or mixed with a florescent probe
       • gel is placed under ultraviolet light and the DNA becomes visable

3. The Southern blot technique

   • used to separate DNA fragments in order to distinguish and identify individuals based on their DNA
   • 1. restriction fragment preparation
   • 2. electrophoresis
   • 3. blot the smear of fragments onto a nitrocellulose filter paper
   • 4. expose paper blot to a solution containing a probe
     • single-stranded DNA complementary to the DNA sequence of interest
     • attaches by base pairing to restriction enzymes of complementary sequence
     • labelled radioactively or with fluroscent dye
   • 5. DNA sequence of interest can be detected by the radioactive or fluroescent dye labelling

4. DNA amplification

   • The polymerase chain reaction (PRC)
     • 1. denature double stranded DNA at 95°C
       • forms single strands
     • 2. cool DNA to 60°C
       • primers are annealed (bonded) to target sequence by complementary base pairing
         • short strands of DNA
         • provide starting and finishing sequence for DNA replication
     • 3. heat DNA to 70°C
       • DNA polymerase initiates DNA replication
     • 4. target sequence can be amplified millions of times
       • Sequencing DNA
         • determining the base code of a specific gene

5. Applications of DNA profling

   • Solving crimes
     • minute samples of DNA can be amplified by PCR
     • compare banding patterns of suspect's DNA and DNA from crime scene
   • Determining paternity
     • each band on a gel of a child's DNA sample has been inherited from either the mother or father
       • father can be identified when his banding pattern is compared to that of the child and mother
   • Sequencing a faulty gene
     • identify base differences in a gene suspected to cause genetic disorder

6. Gene cloning

   • Isolating the gene
     • restriction enzymes
       • probe used to isolate fragment
     • single-stranded DNA primers corresponding to known sequences of gene
       • gene amplified by PRC
   • Manufacturing the product
     • plasmids
       • self-replicating
       • small, circular bacterial DNA molecules
       • human gene is inserted into plasmid
         • plasmid inserted into bacterium
           • genetic transformation
             • introducing DNA into a cell so the DNA is stably maintained within that cell
               • plasmids as vectors
               • bacterophages as vectors
               • YACs and BACs
               • microinjection of DNA into an egg cell
           • bacterial cells selected using antibiotic
             • cells are cultured and produce large quantities of the gene product
         • recombinant DNA- combination of bacterial and human DNA
   • the production of many identical copies of a gene
     • allows manufacturing of large quantities of that gene's product (e.g. insulin)

7. Gene therapy

   • plants
     • more difficult due to presence of cell wall
   • humans
     • replace faulty gene with normal form of gene
     • germ line cells
     • controversial