Genetics 6: Techniques for Producing and Analyzing DNA
Recombinant DNA Technology Scientists can now construct new DNA by combining certain genes with DNA from other areas. Called recombinant DNA Bacteria have restriction enzymes that will cut up invading viral DNA. Scientists can use a special type of restriction enzyme called restriction endonuclease because they cleave double-stranded DNA in the middle of the strand by recognizing a short sequence of nucleotides called the target sequence.
Restriction Enzymes Are specific and predictable (same enzyme will cleave at the same target, the same way, every time) Most produce a staggered cut that leaves unpaired nucleotides at each end called sticky ends Sticky ends can then form base pairs with other single-stranded regions, but only specific ends. If the enzyme produces blunt ends, the piece of DNA can join with any other piece of DNA.
Steps for Making Recombinant DNA A restriction endonuclease is selected that can cut both the DNA fragments that are going to be combined. Each piece of DNA is reacted with the restriction endonuclease to make the fragments. The two cut DNA fragments are incubated with DNA ligase. This will seal the breaks in the DNA by forming covalent and hydogen bonds.
Gene Cloning in Bacteria We can use bacteria to make many copies of a gene for us through gene cloning. To do this: A recombinant DNA molecule made with a plasmid mixed with the gene we want to be copied is made This foreign DNA is taken up by the bacteria during transformation. Usually the cells need to be treated with certain chemicals to make the membranes permeable to the DNA first. Bacterial cells are put on a Petri dish that has growth media on them
Bacterial colonies with the recombinant DNA are identified using special markers. Cells from the recombinant DNA colonies are selected and grown in a liquid culture to produce a large population. The recombinant DNA molecules are isolated and purified from the bacterial cells. A variety of analysis techniques are used to confirm that the correct recombinant DNA molecule has been made.
The Polymerase Chain Reaction (PCR) The process for producing large amounts of DNA for use or analysis is called DNA amplification. Polymerase Chain Reaction is a type of DNA amplification used to amplify the DNA of smaller segments for cloning or analysis purposes. Much faster and highly specific
Steps of PCR: DNA sample is heated to a high temperature (95°C) so the DNA helix denatures into single strands. Then it is cooled (55°C) with two nucleotide primers that are complementary to each 3’ end. (lower temp allows for the annealing of the primers to the DNA) Heated again (72°C) for Taq polymerase (type of DNA polymerase) to work optimally by adding nucleotides to the primers form 5’ to 3’ Taq polymerse was isolated from heat loving bacteria. Repeat 1-3 (denatures, primer annealing, DNA synthesis) several times
Gel Electrophoresis A technique used to separate molecules according to their mass and charge. Used to separate segments of DNA. A gel made of agarose is submerged in a buffer. The gel allows for the DNA to move through. A positive anode lies at one end and a negative cathode at the opposite end. DNA is also negative, so it repels and moves away from that end. Salts in the buffer carry the charge.
Steps in Gel Electrophoresis Before DNA fragments are added to the gel, a blue dye must be added and a ethidium bromide associates with DNA and fluoresces under UV light. Samples of different sized fragments in solution are then added to preformed wells at the one end of the gel. Gel is placed in the buffer and a power source is turned on. DNA fragments start to move. Smaller DNA fragments move more easily through the gel and therefore travel further. Gel is removed from the buffer and exposed to UV light.
DNA Fingerprinting Technology used to identify individuals based on analyzing their DNA sequence Use restriction enzymes to cut pieces of DNA and then separate the DNA through gel electrophoresis. To match suspect DNA with samples found at a crime scene, bands would be the same on the electrophoresis. Called restriction enzyme fragment length polymorphism (RFLP) analysis.
Another form of DNA fingerprinting is called short tandem repeat (STR) profiling. STRs are repeating short sequences of DNA in the genome that vary in length between individuals. Numerous loci can be analyzed (the more analyzed, the more confident the results)
Applications Crime scene investigations DNA found at a crime scene is amplified by PCR, then run through a gel electrophoresis and compared to suspects or victims. Identify victims (September 11th victims) Parental disputes