Biotechnological Tools and Techniques

Restriction Endonucleases (Enzymes) WATCH THIS!!!

Restriction Endonucleases Molecular scissors. Recognizes specific sequence (recognition site) on DNA by disrupting hydrogen bonds between bases. Recognition sites have a complementary palindromic sequence. Recognition sites have a complimentary palindromic sequence…both strands have the same base sequence when read in the 5’ to 3’ direction.

Examples of Palindromic Sequences RACECAR KAYAK MADAM, I’M ADAM A MAN, A PLAN, A CANAL, PANAMA A palindromic sequence is the very same, when read either forwards or backwards!

Restriction Endonucleases Note the palindromic recognition site: Sticky ends are preferred because they can be joined with hydrogen bonds (more eager to establish), whereas blunt ends need to establish covalent bonds to join them (more difficult and needs lots of energy). In this case, “sticky ends” are produced. Some endonucleases produce “blunt ends”.

Restriction Endonucleases Naturally found in bacteria for defense. e.g., Bacteriophage DNA injected into a bacterium is digested into fragments.

Methylases Prokaryotic enzymes that modify palindromic recognition sites by adding methyl groups (-CH3) to one of the bases. Prevents restriction enzymes from cutting the bacterium’s own DNA. Foreign DNA is not methylated, leaving it prone to degradation. “Don’t eat me!”

DNA Ligase Two sticky ends produced with the same endonuclease will naturally bond together. DNA ligase reforms phosphodiester bonds of the backbone. Scientists use T4 DNA ligase to join blunt ends together. WATCH THIS!!! DNA ligase reforms the phosphodiester bond of the backbone via a condensation reaction.

Purpose: DNA fragments separated according to size. Gel Electrophoresis Purpose: DNA fragments separated according to size. DNA subjected to endonucleases are cleaved into fragments of different lengths. DNA mixed with a glycerol-containing dye, allowing greater visualization. DNA loaded into wells. Gel is submerged in an electrolytic solution (buffer). A negative charge is placed near the wells and a positive charge is at the opposite side of the gel. Since nucleotides have the same charge-to-mass ratio, the only difference between DNA fragments is the number of nucleotides.

Gel Electrophoresis

Gel Electrophoresis DNA travels toward positive electrode. Shorter fragments travel faster than longer fragments. Fragments generated with a particular endonuclease produce a characteristic banding pattern. The size of the fragments is determined by using a molecular marker as a standard. The negatively charged DNA travels toward the positive electrode. Shorter fragments travel faster than longer fragments  encounteres less resistance (think of 1 individual running through forest compared to a chain of 20 people…much more difficult and harder to move, therefore slower) The size of the fragments is determined by using a molecular marker as a standard…contains fragments of known size.

Gel Electrophoresis WATCH THIS!!!

Recombinant DNA Restriction enzymes splice foreign genes into plasmids. Recombinant DNA forms and is re-introduced into bacteria.

Recombinant DNA

Transformation (The introduction of DNA from another source) Plasmids are used as vectors to carry desired genes into a host cell. Bacterium that readily takes up foreign DNA is a competent cell. Bacterium can be chemically induced with a CaCl2 solution (at 0ºC) to become competent. A heat shock treatment creates a draft that “sucks” plasmids into the cell. Bacterial plasmids can carry genes for antibiotic resistance!

Transformation WATCH THIS!!!