10. Restriction Endonucleases/ restriction enzymes
- act as scissors and cut specific base-pair sequence known as recognition site
- these enzymes are used to cut double stranded DNA in a predictable and precise manner
- most recognition sites are characterized by a complementary palindromic sequence.
- palindromic = bot strands have same base sequence when read in 5' to 3'
- enzymes bind, disrupts (hydrolysis reaction) the phosphodiester bond btwn two base pairs
- then hydrogen bonds in between the cuts are broken
9. Sticky/ Blunt Ends
- sticky ends= ends of DNA fragment with short s.s. overhangs, resulting from cleavage by restriction enzymes
- blunt ends= fully base paired ends of DNA fragment resulting from cleavage by restriction enzymes
- sticky ends more useful b/c it can be joined more easily to other sticky end that has been produced by the same restriction endonuclease through complementary base pairing
8. Methylases
- enzymes that add methyl group to one of the nucleotides found in a restriction endonuclease recognition site
- prevent the bacteria's immune system to cleave its own DNA
7. DNA Ligase
- glues two fragments of DNA, generated using the same restriction enzyme, together
- two fragments are naturally attracted to each other b/c of their complementary base pairs
- hydrogen bonds will form btwn the nucleotides but it is not a stable arrangement
- phosphodiester linkage must be reformed
- DNA ligase uses condensation reaction and drives out a molecule of water to form phosphodiester bond
6. Gel Electrophoresis
- separation of charged molecules by size in a gel
- DNA -> negatively charged, relatively similar mass nucleotides
- the fragments are put in a gel where one side is +'vely charged and other -'vely
- DNA fragments are attracted to +'ve side and moves towards it
- long fragments don't go that far and small fragments move closer to +'ve side b/c they are small and can navigate through pores
- agarose = polysaccharide found in seaweed used to form gell meshwork
5. Vector Cloning/Transformation
- the fragments of DNA with sticky ends are put into a plasmid of a bacteria
- plasmids are engineered to have multiple-cloning site, a region in plasmid that contain recognition sites of a number of restriction endonucleases
- introduction of foreign DNA, usually by plasmid or virus, into a bacterial cell
- plasmids are vectors
- bacterium that readily take up foreighn DNA is called competent cell
- selective plating isolates the cells with recombinant DNA
- cloned vectors have antibiotic-resistance gene thus if transformation is successful the bacteria will be able to grow on media that contain the antibiotic
4. PCR: Polymerase Chain Reaction
- similar to DNA replication in nucleus but instead of helicase and gyrase, heat is used to separate the strands
- at 94C - 96C the hydrogen bonds are broken
- DNA primers are synthesized in lab and is used to start the elongation
- DNA primers has to be complementary to the target area to be copied
- the temperature is brought down to 50C - 65C to let the primers anneal
- Taq polymerase starts building the complementary starnds at 72C
3. RFLP: Restriction Fragment Length Polymorphism
- polymorphism= difference in DNA sequence,, coding or non-coding, that can be detected btwn individuals
- polymorphism in coding region can be identified with specific mutations. ex. sickle cell
- RFLP= method used to compare differences in DNA fragments btwn individuals using restriction endonucleases
2. DNA Sequencing
- Sanger dideoxy method is most popular
- similar to DNA replication
- need 4 identical single-stranded DNA with radioactively labelled primer in 4 different test tubes
- DNA polymerase and a supply of free nucleotides in form of all four deoxynucleoside triphosphates (dATP, dTTP, dGTP, and dCTP) are added
- each test tube contains dideoxy analogue of one of the deoxynucleoside triphosphates (dNTPs)
- dideoxy analogue stops elongation b/c it does not have -OH group on the 3' carbon to create a phosphodiester bond
- thus, different lengths of complementary DNA is built
- the fragments can be read in a gel
1. Application
- restriction enzymes are expensive thus cannot use many in real life
- complete digestion is also unrealistic
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