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Purpose • Learn about single nucleotide polymorphisms (SNPs) in human DNA. • Describe how SNP analysis can be used to identify disease risks, find genotypes, predict phenotypes and identify ancestral origins. Prepare and present information for your colleagues regarding one SNP and its uses. Learn how to collect and separate DNA from a sample of body fluid. Practice using micropipets and gel electrophoresis equipment Use polymerase chain reaction (PCR) to amplify selected SNP DNA. Analyze amplified DNA fragments using gel electrophoresis. Identify whether you are homozygous or heterozygous for specific SNP markers, and interpret these results.
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Explain a SNP: This portion of the project involves summarizing and presenting information about one particular SNP. Working in groups of 2-3, you conduct the research and prepare a poster describing your SNP. The poster session will be held on the day of your scheduled final exam (Seniors will present their posters on the Friday before finals). Select your SNP from the enclosed “SNP List”, or choose another SNP to study (with prior approval of your instructor). Your final presentation should answer the following questions about your SNP: • • • • What is the SNP “rs” code number and what are the alleles? In which gene, and on which chromosome is it located? What is the gene name/code? How does the SNP variant affect gene function? What condition/attribute does the SNP variant predict? (e.g. ancestral origin, disease risk, phenotype, etc.) • What is the incidence of the SNP variant (population proportion, different geographic distribution, etc). • • • • What is the research correlation between the SNP and the condition/attribute? How rigorous is the correlation? Why is it useful for people to know their genotype for this SNP? Do you think most people would want to know whether they have this SNP? Why or why not?
These types of SNPs can serve as “biological markers” and were used in chromosome mapping during the Human Genome Project.S. sickle-cell anemia). Here are a few of the common uses of SNP DNA analysis: • Specific SNPs are more common in certain populations than others. Most traits derive from the interaction of several genes or a combination of genetic and environmental factors. SNPs occur every 250-1000 base pairs. SNPs within coding regions are of great interest to scientists in the fields of genealogy and medicine. In order to be classified as a SNP. . the U. A few harmful SNPs can directly cause a disease-producing allele (e. offering a genetic profile for a fee of $300-$900.000 different SNPs have been identified. To date. Consumer groups have raised concerns that genetic information could be used to discriminate against individuals who carry certain SNP alleles.000. and several companies offer genetic tests directly to consumers. and comprise approximately 90% of the total genomic variability (Alus and tandem repeats are the other main variable regions).Background Information About SNPs and Genetic Testing SNPs (single nucleotide polymorphisms) are variations in a single location within the human genome. allowing scientists to track human migration patterns or determine ancestral origins. enacted the Genetic Information Nondiscrimination Act (GINA) as a safeguard for the privacy of genetic data. DNA Direct. and give consumers an assessment of their genetic disease risk or information about the alleles they carry. • • • Consumer demand for genetic testing has increased recently. Genelex and Navigenics are examples of genetic testing companies. mitochondrial and Y-chromosome DNA analysis. Medicines don’t work equally well in all people. Analysis of SNPs allows scientists to assess genetic predisposition for certain illnesses. and most are found within non-coding regions of the DNA. approximately 3. Some individuals have adverse responses and some do not respond well. In 2008. Certain SNPs are good predictors of sensitivity/insensitivity to medications. and can allow doctors to prescribe a more personalized effective treatment. the variation must occur in at least 1% of the population. 23andMe. Most of the companies use SNP.g.
and then analyze the class set of data. depending upon genotype. First. we are not studying any “scary SNPs”. Students will study two of these traits in their DNA. so that DNA results will be anonymous.SNP Testing at PALY We will take several steps to ensure the confidentiality of our collective genetic information. we will require that each student and their parent/guardian sign an informed consent form and abide by our privacy provisions. This DNA fragment results from the outer primers and serves as an internal positive control for the PCR procedure. • • . T rait G ene SN P A llele 1 C A llele 2 T Size 1 17 3 Size 2 22 5 O uter Size 34 2 G enotype/Phenotype earwax A B C C 11 rs17 82 29 31 C C or C T = wet T T = dry A A = brown (85% ) A G = brown (56% ). • A non-specific DNA fragment is produced in all individuals. The traits. you will obtain either two or three different amplified DNA segments. green (37% ) G G = blue or green (99% ) T T or T C = can digest milk C C = lactose intolerant C C = sprinter C T = m ixed T T = endurance G G or G C = taster C C = non a taster eye color HERC2 rs12913832 A G 276 210 432 lactose intolerance muscle type LC T rs4988235 C T 135 188 268 ACTN3 rs18 15 73 9 C T 18 9 23 6 37 3 P T C taster T A S2R 38 rs71 35 98 C G 16 0 23 8 34 4 Tetra-primer ARMS-PCR Procedure for SNPs Tetra-primer ARMS-PCR is a widely accepted method for amplifying SNP DNA. Homozygotes will produce one additional fragment resulting from one of the inner primers. but not an indication of genetic fitness or risk for any serious disease. it employs two sets of primers. During the PCR procedure. Unlike the traditional PCR procedure. In addition. our protocol calls for students to use a PIN# rather than their name. The “outer primers” set the boundary for the DNA region containing the SNP. The “inner primers” are specific for each allele. Finally. one from each of the inner primers. We will analyze traits that are of interest. genes and alleles we will use are outlined in the table below. Heterozygotes will produce two additional DNA fragments.
The inner and outer primer arrangement for one particular SNP is shown below. . and individuals will produce that DNA segment only if they carry the specific allele. When the tetra-primer ARMS-PCR procedure is completed. The outer primer pair allows for the non-specific band to be produced. Homozygotes have one non-specific and one specific band (two bands total). the DNA fragments are separated by gel electrophoresis. The PCR products produce a characteristic fragment size pattern: Heterozygous individuals have one non-specific band and two specific bands of known length (3 bands total). Each inner primer (G or A) is allele-specific.
simple. and these impact the way you analyze the gels after electrophoresis. and must be disregarded when analyzing the gel. The outer primers are often non-specific. SNP genotype can therefore be determined by whether the allele-specific band is present or absent. The ladder allows determination of the size of each DNA band. However. 400 O uter band = 344 Inner band=160 N o inner band at 238 H om ozyg ous non-taster Eye color Extraneous band = 600 O uter band = 400 Inner band = 210 N o inner band at 276 H om ozyg ous blue/green L actose tolerance Extraneous bands = 400. lactose tolerance and PTC taster. The ladder standard and known primer fragment sizes together serve as key points of comparison. This results in extraneous bands on the gel. ladder eye lac PTC ladder G el Q uality Lad der has b ands: 2072 1500 1400 1300 1200 1100 1000 900 800 700 600 (b right) 500 400 300 200 100 Ladd er bands are close together Ladder o nly co vers ½ of the gel length B etter result if the gel is run longer P T C T a ste Extraneous band= 600. These bands must be disregarded when determining the individuals SNP genotype. there are some inherent limitations to the methodology. Observe that you can determine the individual’s genotype despite the presence of extraneous DNA bands and primer dimers. • Primer dimers are common because the inner primers are very similar and often adhere to one another. Primer dimers are usually smaller than 100 base pairs. • • Here is a sample gel containing three of the SNPs that we will study: eye color.Limitations of Tetra-Primer Systems Using both primer sets at one time allows for rapid. low-cost analysis of SNP DNA. the size of the outer non-specific and inner allele-specific DNA fragments are known. and may amplify DNA outside the SNP region. In SNP analysis. 500 O uter band = 268 Inner band =135 N o inner band at 188 Primer dimer <100 H omozygous tolerant .
AP Biology SNP List Pre-approved SNPs are listed (information is readily available) Only one group per class may select each category (then select one SNP in that category) C ateg ory SN P rs# C ateg ory SN P rs# Alzheimer’s disease risk rs4420638 rs429358 rs324650 rs1799971 rs6539137 rs4630362 rs10861192 rs7794745 rs2710102 rs1858830 rs1322784 rs1695 D iabetes T ype 2 rs7903146 rs12255372 rs662799 Alcohol reactions Resistance to high dietary fat ALS disease Folate metabolism rs1901133 Autism risk H eart disease risk rs133049 rs7412 rs429358 Asthma risk H IV resistance rs333 Anesthesia sensitivity rs1805007 H air color rs1805007 rs12821256 rs283414 rs34778348 rs34637584 rs1790024 rs10945791 rs45539432 B reast cancer risk rs12255372 rs1799950 rs498650 rs16942 rs1799966 rs766173 rs1801426 rs6152 Parkinson’s disease risk B aldness resistance N icotine dependence rs1051730 rs3750344 rs9939609 Cancer risk (other sites) rs1801133 rs6983267 rs2032582 rs4444903 rs2899109 rs910586 rs1934328 rs332 O besity Cleft palate Pleasure pathways rs1800497 Cystic fibrosis Restless leg syndrome rs6710341(A) rs12469063(G) rs1799853 rs9923231 rs1057910 Crohn’s disease rs17221417 rs6596075 W arfarin sensitivity Cognitive processing rs4680 .
www.org/cgi-perl/gbrowse/hapmap26_836/ Type in the rs#. http://23andme.com Enter your SNP rs#. You can get a gene view and chromosome map including the SNP. • Once you find the gene name/code. .hapmap. • General searches on the genetics of your category (hair color.com You can use the general information in 23andme to get ideas about genetic testing and the reliability of genetic indicators. etc) can also yield useful information. or click on the SNP under “Popular SNPs” to get additional information.Useful Web Sites to find information about your SNP: • http://snpedia. You can find the gene name and chromosome#.gov/projects/SNP Search SNP for (enter rs#). you can type that into a search engine.you can often find interesting information about a particular SNP in that spot as well. restless leg syndrome. and find out the nature of the mutation (missense or nonsense or silent) www.ncbi. • • • Other information gathering strategies: • You can type the rs# for your SNP into any search engine such as Google or Yahoo.nlm. Click on “Medical Condition” or “Genes” buttons on the left side of the webpage to get additional SNPs. Spittoon is a blog associated with 23andme . Use the PubMed link from SNPedia to get additional abstracts and research on your SNP. get a picture of the gene/allele and find interesting data on the frequency of the SNP in various human population.
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