Using an Alu Insertion Polymorphism to Study Human …

1 21-1230 21-1230A 21-1231. 21-1231A 21-1232 21-1232A. Using an Alu Insertion Polymorphism to Study Human Populations Using an Alu Insertion Polymorphism to Study Human Populations IMPORTANT INFORMATION. Storage: Upon receipt of the kit, store proteinase K, PV92B primer/loading dye mix, and DNA marker pBR322/BstNI in a freezer (approximately 20 C). All other materials may be stored at room temperature (approximately 25 C). Use and Lab Safety: The materials supplied are for use with the method described in this kit only. Use of this kit presumes and requires prior knowledge of basic methods of gel electrophoresis and staining of DNA. Individuals should use this kit only in accordance with prudent laboratory safety precautions and under the supervision of a person familiar with such precautions.

2 Use of this kit by unsupervised or improperly supervised individuals could result in injury. Limited License: Polymerase chain reaction (PCR) is protected by patents owned by Hoffman-La Roche, Inc. The purchase price of this product includes a limited, non-transferable license under Patents 4,683,202; 4,683,195; and 4,965,188 or their foreign counterparts, owned by Hoffmann-La Roche Inc. and F. Hoffmann-La Roche Ltd. (Roche), to use only this amount of the product to practice the Polymerase Chain Reaction (PCR) and related processes described in said patents solely for the research, educational, and training activities of the purchaser when this product is used either manually or in conjunction with an authorized thermal cycler.

3 No right to perform or offer commercial services of any kind Using PCR, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at The Perkin-Elmer Corporation, 850 Lincoln Center Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501. Printed material: The student instructions, pages 5 24, as well as the CarolinaBLU staining protocol on page 32 may be photocopied as needed for use by your students. DNA KITS. Learning Center Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory.

4 All rights reserved. REAGENTS, SUPPLIES, AND EQUIPMENT CHECKLIST. Included in the kit: Needed but not supplied: DNA extraction and amplification (all kits): saline solution (NaCl), 10 mL per g Chelex resin student, in 15-mL tube 5 mL proteinase K (100 g/mL) Micropipets and tips (1 L to 1000 L). 700 L PV92B primer/loading dye mix microcentrifuge tubes, polypropylene, 25 *Ready-to-Go PCR Beads 2 per student 5 mL mineral oil Microcentrifuge tube racks 130- L tube pBR322/BstNI markers Microcentrifuge for tubes ( g/ L) or PCR tubes, 1 per student Instructor's manual with reproducible ( microcentrifuge tubes may also be used.). Student Lab Instructions or tube adapters for Alu CD-ROM microcentrifuge (can be made from and/or tubes).

5 ** electrophoresis kits with ethidium bromide staining Thermal cycler, programmable (Kits 21-1231and 21-1231A) also include: electrophoresis chambers 5 g agarose electrophoresis power supplies 150 mL 20 TBE Gel-staining trays 250 mL ethidium bromide, 1 g/mL UV transilluminator (ethidium bromide 4 latex gloves staining). 6 staining trays White light box (CarolinaBLU staining, optional). ** electrophoresis kits with CarolinaBLU staining Camera or photo-documentary system (optional).. (Kits 21-1232 and 21-1232A) also include: 5 g agarose Paper cup, 1 per student . 150 mL 20 TBE. Permanent markers . 7 mL CarolinaBLU Gel & Buffer Stain Container with cracked or crushed ice . 250 mL CarolinaBLU Final Stain Boiling water bath (optional, see instructions).

6 4 latex gloves 6 staining trays *Ready-to-Go PCR Beads incorporate Taq polymerase, dNTPs, and MgCl2. Each bead is supplied in an individual tube or a tube. ** electrophoresis reagents must be purchased separately for Kits 21-1230 and 21-1230A. DNA KITS. Learning Center Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. Using an Alu Insertion Polymorphism to Study Human Populations CONTENTS. STUDENT LAB INSTRUCTIONS ..5. INTRODUCTION ..5. LAB FLOW ..7. METHODS ..8. BIOINFORMATICS ..13. RESULTS AND DISCUSSION ..17. INFORMATION FOR INSTRUCTOR ..25. CONCEPTS AND METHODS ..25. LAB SAFETY ..25. INFORMED CONSENT AND DISCLOSURE ..26. INSTRUCTOR PLANNING, PREPARATION, AND LAB FINE POINTS.

7 26. CarolinaBLU STAINING ..32. BIOINFORMATICS ..33. ANSWERS TO BIOINFORMATICS QUESTIONS ..33. ANSWERS TO DISCUSSION QUESTIONS ..34. CD-ROM CONTENTS ..36. DNA KITS. Learning Center Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. 5. STUDENT LAB INSTRUCTIONS. INTRODUCTION. Although DNA from any two people is more alike than different, many chromosome regions exhibit sequence differences between individuals. Such variable sequences are termed polymorphic (meaning many forms). and are used in the Study of Human evolution, as well as for disease and identity testing. Many polymorphisms are located in the estimated 98% of the Human genome that does not encode protein. This experiment examines a Polymorphism in the Human genome that is caused by the Insertion of an Alu transposon, or transposable element.

8 Alu is a member of the family of short interspersed elements (SINEs) and is approximately 300 nucleotides in length. Alu owes its name to a recognition site for the endonuclease AluI in its middle. Although Alu is sometimes called a jumping gene, it is not properly a gene, because it does not produce a protein product. Alu transposons are found only in primate genomes and have accumulated in large numbers since primates diverged from other mammals. Human chromosomes contain more than one million Alu copies, equaling about 10% of the genome by mass. This accumulation was made possible by a transposition mechanism that reverse transcribes Alu mRNAs into mobile DNA copies. Another transposon, the long interspersed element (LINE) L1, supplies a specialized reverse transcriptase enzyme needed for Alu to jump.

9 Hence, Alu and L1 exist in a sort of molecular symbiosis. At any point in evolutionary time, only one or several Alu masters were capable of transposing. Although the rate of transposition was once much higher, a new Alu jump is estimated to now occur once per 200 live Human births. There is lively debate about whether Alu serves some larger purpose in primate genomes or is merely selfish DNA that has been successful in its mode of replication. Alu insertions in coding exons are implicated in a number of Human diseases, including neurofibromatosis, thalassemia, cancer, and heart attack. However, the vast majority of Alus are located in introns or intergenic regions, where they appear to have no phenotypic effect. Alus in introns have had a potentially important impact on protein evolution: they provide alternative splice sites in approximately 5% of genes that produce multiple protein products.

10 Each Alu is the fossil of a unique transposition event that occurred once in primate history. After the initial jump, an Alu is inherited from parents by offspring in a Mendelian fashion. The vast majority of Alu insertions occurred millions of years ago and are fixed. This means that, for a particular locus, all primates have inherited Alus on each of the paired chromosomes. However, several thousand Alus have inserted in our genome since humans branched from other primates. Some of these are not fixed, meaning the Alu Insertion may be present or absent on each of the paired Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. 6 Using an Alu Insertion Polymorphism to Study Human Populations chromosomes, thus creating two possible alleles (+ and ).