AP Biology Project Snip Out Your SNPs!

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.

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?

Background Information About SNPs and Genetic Testing SNPs (single nucleotide polymorphisms) are variations in a single location within the human genome,

and comprise approximately 90% of the total genomic variability (Alus and tandem repeats are the other main variable regions). In order to be classified as a SNP, the variation must occur in at least 1% of the population. To date, approximately 3,000,000 different SNPs have been identified. SNPs occur every 250-1000 base pairs, and most are found within non-coding regions of the DNA. These types of SNPs can serve as “biological markers” and were used in chromosome mapping during the Human Genome Project.

SNPs within coding regions are of great interest to scientists in the fields of genealogy and medicine.

Here are a few of the common uses of SNP DNA analysis: • Specific SNPs are more common in certain populations than others, allowing scientists to track human

migration patterns or determine ancestral origins. • A few harmful SNPs can directly cause a disease-producing allele (e.g. sickle-cell anemia).

• Most traits derive from the interaction of several genes or a combination of genetic and environmental

factors. Analysis of SNPs allows scientists to assess genetic predisposition for certain illnesses. • Medicines don’t work equally well in all people. Some individuals have adverse responses and some do

not respond well. Certain SNPs are good predictors of sensitivity/insensitivity to medications, and can allow doctors to prescribe a more personalized effective treatment.

Consumer demand for genetic testing has increased recently, and several companies offer genetic tests

directly to consumers. Most of the companies use SNP, mitochondrial and Y-chromosome DNA analysis, and give consumers an assessment of their genetic disease risk or information about the alleles they carry. 23andMe, DNA Direct, Genelex and Navigenics are examples of genetic testing companies, offering a genetic profile for a fee of $300-$900. Consumer groups have raised concerns that genetic information could be used to discriminate against individuals who carry certain SNP alleles. In 2008, the U.S. enacted the Genetic Information Nondiscrimination Act (GINA) as a safeguard for the privacy of genetic data.

SNP Testing at PALY

We will take several steps to ensure the confidentiality of our collective genetic information. First, we are not studying any “scary SNPs”. We will analyze traits that are of interest, but not an indication of genetic fitness or risk for any serious disease. In addition, we will require that each student and their parent/guardian sign an informed consent form and abide by our privacy provisions. Finally, our protocol calls for students to use a PIN# rather than their name, so that DNA results will be anonymous.

The traits, genes and alleles we will use are outlined in the table below. Students will study two of

these traits in their DNA, and then analyze the class set of data.

T rait

G ene

SN P

A llele 1

A llele 2

Size 1

Size 2

O uter Size

G enotype/Phenotype

earwax

A B C C 11

rs17 82 29 31

C

T

17 3

22 5

34 2

C C or C T = wet T T = dry

eye color

H E R C 2

rs12913832

A

G

276

210

432

A A = brown (85%) A G = brown (56%), green (37%) G G = blue or green (99%)

lactose intolerance

LC T

rs4988235

C

T

135

188

268

T T or T C = can digest milk C C = lactose intolerant

muscle type

A C T N 3

rs18 15 73 9

C

T

18 9

23 6

37 3

C C = sprinter C T = m ixed T T = endurance

P T C taster

T A S2R 38

rs71 35 98

C

G

16 0

23 8

34 4

G G or G C = taster C C = non a taster

Tetra-primer ARMS-PCR Procedure for SNPs

Tetra-primer ARMS-PCR is a widely accepted method for amplifying SNP DNA. Unlike the traditional PCR procedure, it employs two sets of primers. The “outer primers” set the boundary for the DNA region containing the SNP. The “inner primers” are specific for each allele. During the PCR procedure, you will obtain either two or three different amplified DNA segments, depending upon genotype.

• A non-specific DNA fragment is produced in all individuals. This DNA fragment results from the

outer primers and serves as an internal positive control for the PCR procedure. • Homozygotes will produce one additional fragment resulting from one of the inner primers.

• Heterozygotes will produce two additional DNA fragments, one from each of the inner primers.

The inner and outer primer arrangement for one particular SNP is shown below. The outer primer pair allows for the non-specific band to be produced. Each inner primer (G or A) is allele-specific, and individuals will produce that DNA segment only if they carry the specific allele.

When the tetra-primer ARMS-PCR procedure is completed, 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). Homozygotes have one non-specific and one specific band (two bands total).

Limitations of Tetra-Primer Systems

Using both primer sets at one time allows for rapid, simple, low-cost analysis of SNP DNA. However, there are some inherent limitations to the methodology, and these impact the way you analyze the gels after electrophoresis.

• 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, and must be disregarded when analyzing the gel. • The outer primers are often non-specific, and may amplify DNA outside the SNP region. This results

in extraneous bands on the gel. These bands must be disregarded when determining the individuals SNP genotype.

• The ladder standard and known primer fragment sizes together serve as key points of

comparison. In SNP analysis, the size of the outer non-specific and inner allele-specific DNA fragments are known. The ladder allows determination of the size of each DNA band. SNP genotype can therefore be determined by whether the allele-specific band is present or absent.

Here is a sample gel containing three of the SNPs that we will study: eye color, lactose tolerance and

PTC taster. Observe that you can determine the individual’s genotype despite the presence of extraneous DNA bands and primer dimers.

ladder eye lac PTC ladder

Lad der has b ands: 2072 1500 1400 1300 1200 1100 1000 900 800 700 600 (b right) 500 400 300 200 100

G el Q uality

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, 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, 500 O uter band = 268 Inner band =135 N o inner band at 188 Primer dimer <100

H omozygous tolerant

C ateg ory

SN P rs#

D iabetes T ype 2

rs7903146 rs12255372

Resistance to high dietary fat

rs662799

Folate metabolism

rs1901133

H eart disease risk

rs133049 rs7412 rs429358

H IV resistance

rs333

H air color

rs1805007 rs12821256

Parkinson’s disease risk

rs283414 rs34778348 rs34637584 rs1790024 rs10945791 rs45539432

N icotine dependence

rs1051730 rs3750344

O besity

rs9939609

Pleasure pathways

rs1800497

Restless leg syndrome

rs6710341(A) rs12469063(G)

W arfarin sensitivity

rs1799853 rs9923231 rs1057910

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#

Alzheimer’s disease risk

rs4420638 rs429358

Alcohol reactions

rs324650 rs1799971

ALS disease

rs6539137 rs4630362 rs10861192

Autism risk

rs7794745 rs2710102 rs1858830 rs1322784

Asthma risk

rs1695

Anesthesia sensitivity

rs1805007

B reast cancer risk

rs12255372 rs1799950 rs498650 rs16942 rs1799966 rs766173 rs1801426

B aldness resistance

rs6152

Cancer risk (other sites)

rs1801133 rs6983267 rs2032582 rs4444903

Cleft palate

rs2899109 rs910586 rs1934328

Cystic fibrosis

rs332

Crohn’s disease

rs17221417 rs6596075

Cognitive processing

rs4680

Useful Web Sites to find information about your SNP: • http://snpedia.com

Enter your SNP rs#, or click on the SNP under “Popular SNPs” to get additional information. Click on “Medical Condition” or “Genes” buttons on the left side of the webpage to get additional SNPs. Use the PubMed link from SNPedia to get additional abstracts and research on your SNP.

• http://23andme.com

You can use the general information in 23andme to get ideas about genetic testing and the reliability of genetic indicators. Spittoon is a blog associated with 23andme - you can often find interesting information about a particular SNP in that spot as well.

• www.ncbi.nlm.gov/projects/SNP

Search SNP for (enter rs#). You can get a gene view and chromosome map including the SNP, and find out the nature of the mutation (missense or nonsense or silent)

• www.hapmap.org/cgi-perl/gbrowse/hapmap26_836/

Type in the rs#. You can find the gene name and chromosome#, get a picture of the gene/allele and find interesting data on the frequency of the SNP in various human population.

Other information gathering strategies: • You can type the rs# for your SNP into any search engine such as Google or Yahoo. • Once you find the gene name/code, you can type that into a search engine. • General searches on the genetics of your category (hair color, restless leg syndrome, etc) can also yield

useful information.