Molecular Tools and Techniques

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Mind Map on Molecular Tools and Techniques, created by lt2012723 on 07/07/2014.
lt2012723
Mind Map by lt2012723, updated more than 1 year ago
lt2012723
Created by lt2012723 almost 11 years ago
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Molecular Tools and Techniques
  1. Restriction Enzymes
    1. bacterial enzyme
      1. recognise a short sequence of bases in a DNA molecule
        1. cuts the DNA at this recognition site
          1. 'molecular scissors'
            1. blunt ends
              1. do not ligate
              2. sticky ends
                1. staggered cut
                  1. short, single-stranded ends
                  2. DNA molecule with sticky ends another DNA molecule with complementary sticky ends
                    1. two pieces join up
                      1. reform hydrogen bonds
                        1. DNA ligase stabilises recombined molecule
                          1. 'molecular glue'
                  3. different restriction enzymes recognise different sites
                    1. cut the DNA into different sized fragments
                      1. unique banding pattern on a gel
                        1. gives DNA profile of that individual
                2. Gel Electrophoresis
                  1. DNA is cut into many fragments of different lengths by restriction enzymes
                    1. fragments are sequenced according to size and charge
                    2. fragments are placed into the wells of an agarose gel
                      1. electric field is set up
                        1. negatively charged DNA fragments are drawn to the positive terminal
                        2. highly porous, acts like a sieve
                          1. shortest DNA fragments are able to move more quickly and easily through pores
                            1. travel the furthest distance
                            2. more difficult for longer fragments to pass through pores
                              1. lag behind shorter fragments
                            3. gel is injected with a stain or mixed with a florescent probe
                              1. gel is placed under ultraviolet light and the DNA becomes visable
                          2. The Southern blot technique
                            1. used to separate DNA fragments in order to distinguish and identify individuals based on their DNA
                              1. 1. restriction fragment preparation
                                1. 2. electrophoresis
                                  1. 3. blot the smear of fragments onto a nitrocellulose filter paper
                                    1. 4. expose paper blot to a solution containing a probe
                                      1. single-stranded DNA complementary to the DNA sequence of interest
                                        1. attaches by base pairing to restriction enzymes of complementary sequence
                                          1. labelled radioactively or with fluroscent dye
                                          2. 5. DNA sequence of interest can be detected by the radioactive or fluroescent dye labelling
                                          3. DNA amplification
                                            1. The polymerase chain reaction (PRC)
                                              1. 1. denature double stranded DNA at 95°C
                                                1. forms single strands
                                                2. 2. cool DNA to 60°C
                                                  1. primers are annealed (bonded) to target sequence by complementary base pairing
                                                    1. short strands of DNA
                                                      1. provide starting and finishing sequence for DNA replication
                                                    2. 3. heat DNA to 70°C
                                                      1. DNA polymerase initiates DNA replication
                                                      2. 4. target sequence can be amplified millions of times
                                                        1. Sequencing DNA
                                                          1. determining the base code of a specific gene
                                                    3. Applications of DNA profling
                                                      1. Solving crimes
                                                        1. minute samples of DNA can be amplified by PCR
                                                          1. compare banding patterns of suspect's DNA and DNA from crime scene
                                                          2. Determining paternity
                                                            1. each band on a gel of a child's DNA sample has been inherited from either the mother or father
                                                              1. father can be identified when his banding pattern is compared to that of the child and mother
                                                            2. Sequencing a faulty gene
                                                              1. identify base differences in a gene suspected to cause genetic disorder
                                                            3. Gene cloning
                                                              1. Isolating the gene
                                                                1. restriction enzymes
                                                                  1. probe used to isolate fragment
                                                                  2. single-stranded DNA primers corresponding to known sequences of gene
                                                                    1. gene amplified by PRC
                                                                  3. Manufacturing the product
                                                                    1. plasmids
                                                                      1. self-replicating
                                                                        1. small, circular bacterial DNA molecules
                                                                          1. human gene is inserted into plasmid
                                                                            1. plasmid inserted into bacterium
                                                                              1. genetic transformation
                                                                                1. introducing DNA into a cell so the DNA is stably maintained within that cell
                                                                                  1. plasmids as vectors
                                                                                    1. bacterophages as vectors
                                                                                      1. YACs and BACs
                                                                                        1. microinjection of DNA into an egg cell
                                                                                      2. bacterial cells selected using antibiotic
                                                                                        1. cells are cultured and produce large quantities of the gene product
                                                                                      3. recombinant DNA- combination of bacterial and human DNA
                                                                                  2. the production of many identical copies of a gene
                                                                                    1. allows manufacturing of large quantities of that gene's product (e.g. insulin)
                                                                                  3. Gene therapy
                                                                                    1. plants
                                                                                      1. more difficult due to presence of cell wall
                                                                                      2. humans
                                                                                        1. replace faulty gene with normal form of gene
                                                                                          1. germ line cells
                                                                                            1. controversial
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