Genetic Engineering And Biotechnology:IB Syllabus 4.4.1-3 Jen Yau Period 1 TanviJayaraman Jeff Leibenhaut
4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA
What is PCR? Polymerase Chain Reaction Process in which the original molecule or molecules of DNA (the “template”) are replicated by the DNA polymerase enzyme, doubling the number of DNA molecules DNA polymerase enzymes add complimentary deoxynucleotides to the original piece of DNA (dNTPs) Most PCRs use the enzyme Taq polymerase since it is stable under high temperatures Primers – used as a starting point for the polymerase; usually manmade Able to amplify a single piece of DNA, or a small amount of DNA, into millions of copies of the original Widely used in molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, hereditary studies, paternity testing, and many more
Steps of PCR Step 1: Denaturation of the DNA strand in the mixture containing the DNA template, polymerase enzyme, primers, and dNTPs at 95 oC Breaks the hydrogen bonds that hold the target DNA together Step 2: Annealing at 60 oC Allows the primers to form hydrogen bonds (anneal) with their complementary sequences in the target DNA (A – T, C – G) Step 3: Extension/Polymerization at 72 oC PCR polymerase adds dNTP’s from 5’ to 3’, reading the template from 3’ to 5’ side, to make two double stranded molecules Entire cycle is repeated 30-40 times
What is PCR Used for? Microbiology and molecular biology: used in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology Aids in the diagnosis of microbial infections and epidemiological studies Forensics: Genetic profiling Is especially useful because only a tiny amount of original DNA is required to perform the process
4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size
What is Gel Electrophoresis? Electrophoresis: a technique used to separate and sometimes purify macromolecules, especially proteins and nucleic acids, that differ in size, charge or conformation one of the most widely-used techniques in biochemistry and molecular biology. Differences in the molecular weight and solubility of the molecules caused the molecules to rise up the paper at different rates eventually resulting in their separation in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. Easy Analogy: If the gel is a sponge, you can imagine how large and small particles would pass through a sponge at different rates according to particle size/shape versus the size of the holes in the sponge. The rate of molecular movement depends upon molecule size/shape and the direction of movement depends on the electric charge (+ or -) of the molecules, and the density of the gel through which the particle moves
Types of Gel Electrophoresis Proteins and nucleic acids are electrophoresed within a matrix or "gel” The gel is cast in the shape of a thin slab, with wells for loading the sample The gel is immersed in an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular molecules:
Cont. Agarose: a polysaccharide extracted from seaweed. typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. non-toxic. Agarose gels have a large range of separation, but low resolving power By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques Polyacrylamide: a cross-linked polymer of acrylamide. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders) Acrylamide is a potent neurotoxin and should be handled with care Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide. Polyacrylamide gels have a rather small range of separation, but very high resolving power. In DNA, polyacrylamide is used for separating fragments of less than about 500 bp. under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.
4.4.3 State that gel electrophoresis of DNA is used in DNA profiling
DNA Profiling DNA Profiling: The analysis of DNA to identify the organism that it belongs to Gel electrophoresis separates segments of DNA by size and content The pattern created by the different lengths of DNA segments in gel electrophoresis is the DNA profile or “fingerprint”
STRs Gel electrophoresis specifically isolates non-coding DNA Within non-coding DNA are STRS STRs (short tandem repeats) are repeated sequences of nucleotides inherited from parents The number of repeats is shows by the different lengths of DNA segments An individual’s STRs are unique (excluding identical twins) ACGCTAGCATCGTACGTACGTACGTACGTACGTAAATGACATGG
Works Cited Campbell and Reece, Neil Jane. Biology 8th Edition. Pearson: San Francisco 2008. "DNA Profiling." Biotechnology Online. Australia. Web. 24 Feb. 2011. <http://www.biotechnologyonline.gov.au/human/dnaprofile.html>. "PCR. A Review of PCR and Polymerase Chain Reaction." Microbiology Books, Molecular Biology Books, Virology Books, PCR Books. Web. 18 Feb. 2011. <http://www.horizonpress.com/pcr/>. Phillips, Theresa. "Polymerase Chain Reaction - PCR - How Pcr Works." Biotechnology - Biomedical - Biotech. Web. 18 Feb. 2011. <http://biotech.about.com/od/pcr/a/PCRtheory.htm>. "The Polymerase Chain Reaction." Sumanas, Inc. 2006. Web. 18 Feb. 2011. <http://www.sumanasinc.com/webcontent/animations/content/pcr.html>.