Presentation on theme: "Introduction to Techniques"— Presentation transcript:
1 Introduction to Techniques Restriction Enzymes, PCR and Gel Electrophoresis
2 Restriction EnzymesRestriction enzymes are also called restriction endonucleasesThey cut double stranded DNA at sequence specific sitesThey can produce “sticky ends” or blunt ends depending on the enzymeSticky EndsBlunt EndsSticky EndsBlunt Ends
3 Restriction Enzymes1978 Nobel Prize in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleasesRestriction enzymes were discovered in E.coli as a defense mechanism against bacterial viruses (bacteriophages)The recognition sites are usually 4-12 nucleotides longSequences are palindromic (GAATTC)There are hundreds of restriction enzymes currently being used
4 Restriction Enzymes: Applications Restriction enzymes are commonly used in laboratories to create recombinant DNAHarvest DNA products for other applicationsDNase a general nuclease used to eliminate DNA in RNA samples
5 Restriction EnzymesWhat is better for making recombinant DNA: Sticky ends or blunt ends?
8 Restriction Enzymes Student activity Potential Problems: Wrong buffer* activityOnline resourcesClick on TechniquesRestriction enzymes are found in cutting and pasting
9 Gel ElectrophoresisGel electrophoresis is used to separate nucleic acids (DNA and RNA) or proteins for analytical useDNA and RNA are separated using agaroseProteins are separated using polyacrylamideThe gel is a matrix (cross-linked polymers) that allow products to be separatedSeparation is based on the size (not shape) of a product as it moves through a charged fieldWhat is the charge on DNA and proteins? What is the shape? Why is this important?How would you connect the electrodes?
10 Gel ElectrophoresisThe negative charge is at the top (closest to the samples) and the positive charge is at the bottomSamples are negatively charged and will travel towards the positive chargeDNA and RNA are negative because of their sugar-phosphate backboneProteins are denatured to give a constant shape and given a charge through the negative loading buffer usedSamples are diluted in a loading buffer that helps the samples stay in the wells
18 Gel Electrophoresis Online Resources: http://www.dnai.org/b/index.html Potential ProblemsConnecting the charges backwardNot enough loading dyeRunning the gel too hotHandling EtBrOnline Resources:Click on TechniquesGel electrophoresis is found in sorting and sequencing
19 PCRInvented in 1983 by Kary Mullis (Nobel Prize in 1993 for its discovery)Uses primers to exponentially amplify a specific region of DNAComponents needed for the reaction:DNAPrimers to region of interestDNA polymerase (Taq – used to synthesize the DNA)dNTPS (the building blocks of the copied DNA)Buffer (with appropriate salts to ensure the enzyme works properly)
20 PCR Three steps of the reaction: Denaturation: High heat (94-98o) to separate the strands of DNAAnnealing: (50-60o – depends on the primers) this step allows the primers to bind to the denatured DNA strandsElongation (74o) – DNA polymerase synthesizes the new strandThis step is dependant on the length of the product to be amplified (1min/1kb of DNA)Check products with gel electrophoresis and sequencing
24 PCR: Applications Used to test for gene products for disease diagnosis Used to amplify small amounts of materialForensicsFossil RecordsUsed for recombinant DNA technologyUsed for virus detection
25 Resources Potential Problems No amplification due to wrong buffer conditionsNo amplification due to lost enzyme activityPrimers are wrongOnline Resources:Click on TechniquesPCR is found in amplifying