Download SNP Array Activity Learning Objectives Introduction

Biology 362 – Fall 2011
Name ____________________________
SNP Array Activity
Learning Objectives
By the end of this lab, you should be able to
explain how a SNP array is constructed.
describe how a SNP array is used.
compare and contrast SNP arrays and microarrays.
interpret a SNP array and draw conclusions about what alleles an individual has.
explain applications of SNP arrays in health and other fields, using examples.
Introduction
What is a SNP array? How is it similar to a microarray? How is it different? Both of these techniques
have entered widespread use in the wake of the Human Genome Project and improvements in
sequencing technology.
DNA Microarrays
DNA microarrays (also called DNA arrays and gene chips) are manufactured by placing many singlestranded DNA molecules with a single known sequence in a single spot on a glass plate or slide. Many
different sequences may be included in a single microarray, with each sequence being assigned to its
own spot on the microarray. They are often used to look at patterns of gene expression. How? By
extracting mRNA from cells, researchers can isolate what genes are turned “on” – expressed. This
mRNA can be used to make cDNA (complementary DNA) that is fluorescently labeled. The cDNA from
one cell or cell type can be labeled with green; the cDNA from another cell or cell type can be labeled
with red. Both cDNA sets are then added to the slide, allowed to hybridize with any DNA on the slide,
and then washed off. The fluorescent tags can then be “read” (a computer detects the fluorescence and translates this into data output), detecting which cells expressed which genes. These sorts of arrays,
also called expression arrays, can be used to answer a diverse set of questions – what genes are turned
on that initiate a cell’s entry into S phase of the cell cycle? into mitosis? What genes are turned on or off in cancerous cells compared to normal, healthy cells? What genes are turned on that cause the heatshock response in bacteria?
A paper published in 1999 by Eric Lander first illustrated the utility of gene expression arrays. Lander
and his colleagues used gene expression patterns from 6,800 human genes to determine whether a
patient had acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). This rapidly
decreased diagnosis time and expense and sped the entry of patients into the appropriate treatment.
SNP Arrays
Recall that SNP stands for “single nucleotide variation.” In any population, individuals differ in which alleles they have for any give gene. They also differ in small variations – SNPs. These arrays are made by
attaching all the possible variations (alleles) of a gene or several genes in a grid pattern, similarly to
microarray construction. SNPs that are not actually part of a known gene may also be included if the
SNP in question is thought to be associated with a gene or condition of interest or if the researchers are
interested in using the SNP for other reasons.
Introduction modified from http://www.hhmi.org/biointeractive/genomics/microarray.html – “The Basics of DNA
Microarray” by Laura Bonetta
DNA is obtained from a subject and extracted. Multiple copies of the gene or genes of interest are
synthesized with detectable tags (often fluorescent). The copies are then applied to the array and
allowed to hybridize with the DNA on the array. Any non-hybridized DNA is washed away, and the
microarray is “read” by computer, similarly to a microarray. In this case, the array provides information about what sequence variants are present in the individual in question. These arrays can be used to
answer basic biological questions, such as what level of genetic variation is present in a population for a
gene of interest? How much variation is there in the human genome? They can also be used to answer
specific questions, such as whether an individual has the genetic variation or alleles associated with a
particular disease or with responsiveness to a particular drug treatment.
Why are many copies of the gene or genes of interest needed?
How would you synthesize many copies of the gene or genes of interest? Explain how you would do
this.
Other Arrays
Still other techniques include microarray comparative genomic hybridization (microarray CGH), which
identifies deletions and amplifications (of copy number) of genomic regions. These types of changes are
also useful for answering basic biological questions and for detecting increased risk of certain diseases
such as cancer. Other types of arrays can detect modified regions of the genome (methylated or
acetylated regions). Protein arrays are a different approach that is nonetheless conceptually similar to
the above arrays – these arrays use techniques to find specific proteins from a sample. These sorts of
arrays typically are arrays of antibodies arranged on a glass or plastic slide. The antibodies bind to their
specific protein, which can then be detected by adding additional tagged antibodies that bind to the
bound protein or by tagging the proteins before adding them to the array. Other types of protein
arrays bind the proteins to the slides and then look at their activity. Molecules that the proteins of
interest act upon are then added to the array, and the proteins that interact with the molecules can be
detected and identified.
Today’s Lab
Today, we will focus on SNP arrays. These are the types of arrays used by many of the biotech
companies that offer individualized genetic screening. They generate their results by looking at which
genetic variants the individual has and whether the variant the individual has is associated with
particular diseases. You can imagine that this can be somewhat challenging, as many genetic diseases
are not simple Mendelian traits, but complex quantitative traits that are affected by environmental
factors and GxE interactions as well. You will construct a simulated SNP array and then use it to
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determine if an individual has elevated risks for any of the genetic diseases based on the information
from the SNP array.
Materials
SNP array slide (paper)
sequenced genes to use in the manufacture of the SNP chip
DNA of the person of interest
fluorescent ddNTPs
DNA polymerase
Instructions
You will need to determine what, if any, disease risks the individual you are testing has.
First, you need to create your array. You will use the known DNA sequences to decide on the DNA
sequences for the probes. Write the probe sequences in the spots on your array to make a SNP chip you
can use to genotype people.
Once you have your probe sequences on your array, fragment your sample DNA and wash it over the
array, allowing it to hybridize with the DNA on the array.
After the DNA hybridizes, DNA polymerase and fluorescently tagged ddNTPs can be added to elongate
each fragment by one nucleotide.
You would then scan the SNP array and have the computer record which ddNTPs were present for which
spots on the array. This is the information you would use to determine the genotype of the individual.
For this sample, you can cheat a little and read the sequence rather than rely on the fluorescent tags.
That’s not the case with a real SNP array, however. In a real SNP array, why are the fluorescent tags on the ddNTPs necessary?
Questions
1. You’ve been hired after graduation by 23 and me as a technician. One day, you mistakenly add only ddNTPs to the PCR reaction you’re carrying out to amplify a client’s DNA. What will the consequence of this be for the amplification, if any? Explain. What will the consequences of this be for the client, if any?
2. In another reaction, you mistakenly add only dNTPs and no ddNTPs to a SNP array that has a client’s DNA fragments hybridized to it. What will the consequence of this be for your ability to determine what SNPs the client
has and what diseases s/he may be at risk for, if any? Explain. After your previous mistake and this one, have your
chances of becoming “employee of the month” gone up or down? 3. Suppose that you obtain the following results for a SNP array.
4. PCR has become so common that even most introductory biology students have a chance to prepare and run
PCR. In introductory courses, it is common for students to think that PCR is an end in itself: that running PCR might
be the “point” of a DNA extraction and the final step in an experiment. State why this is incorrect, then explain 3
what the point of PCR is and what it can be used for clearly enough that a confused introductory biology student
could be set straight.
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Questions)–)Stickleback)video) )
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Name:__________________________)
Learning!goals:!
• Learn!how!to!find!genes!important!for!adaptive!differences!between!
populations!
• Learn!how!to!functionally!show!that!a!mutation!is!causal!(causes!the!change!
in!phenotype)!
• Learn!about!gene!expression!on/off!switches!
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I!strongly!recommend!re?watching!minutes!4:20!through!10:43!when!answering!
these!questions.!
https://www.youtube.com/watch?v=Z38QHLaGwWM!
OR!
http://www.hhmi.org/biointeractive/making?fittest?evolving?switches?evolving?
bodies!
These!videos!may!also!be!useful!to!rewatch:!
http://www.hhmi.org/biointeractive/gene?switch!
http://www.hhmi.org/biointeractive/pitx1?expression!
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10!points/question!
1) How!did!researchers!determine!that!the!chromosomal!region!containing!the!
gene!Pitx1!is!important!for!the!production!of!pelvic!spines!in!sticklebacks?!(ie!
what!crosses!were!made,!and!what!did!they!do!with!the!progeny)!
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2) How!did!they!figure!out!where!the!Pitx1!gene!is!expressed!in!developing!
sticklebacks?!
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3) Are!differences!in!the!coding!region!of!Pitx1!responsible!for!the!pelvic!spine!
differences!between!ocean!and!lake!sticklebacks?!How!do!you!know!this?!
4) What!type!of!mutation!is!responsible!for!the!pelvic!spine!differences!between!
ocean!and!lake!sticklebacks?!!How!did!they!prove!this?!(hint:!it!involved!
green!fluorescent!protein)!
5) How!did!they!prove!that!this!DNA!region!was!an!on/off!“switch”!for!pelvic!
spine!production?!
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6) When!comparing!ocean!and!lake!sticklebacks,!the!expression!of!this!gene!
differs!in!pelvic!spines,!but!not!in!the!lips.!!How!is!this!possible?!
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2!
Biology'260'
'
Name'____________________________'
Polygenic*Inheritance*
*
Learning*Objectives*
By'the'end'of'this'lab,'you'should'be'able'to'
• explain'how'polygenic'inheritance'works.'
• explain'why'the'inheritance'of'polygenic'traits'is'more'difficult'to'follow'than'simple'Mendelian'
traits.'
• summarize'how'the'environment'and'the'genes'contribute'to'the'inheritance'of'many'polygenic'
traits.'
• understand'the'basic'model'of'polygenic'inheritance:'there'are'many'genes'for'a'trait,'each'with'
at'least'one'allele.''Having'more'of'the'alleles'that'contribute'to'the'disease'increases'the'risk'of'
developing'the'disease.'
• explain'why'a'person'who'has'alleles'that'predispose'them'to'developing'a'disease'and'has'an'
environment'that'predisposes'them'to'a'disease'is'not'guaranteed'to'get'the'disease,'but'does'
have'an'increased'probability'of'developing'the'disease.'
• explain'why'a'person'who'has'alleles'that'predispose'them'to'NOT'developing'a'disease'and'has'
an'environment'that'does'NOT'predispose'them'to'developing'a'disease'can'still'develop'the'
disease'despite'the'lower'probability'of'the'disease'developing.'
• Be'able'to'develop'a'basic'simulation'of'polygenic'inheritance'and'environmental'effects'to'
draw'conclusions'about'disease'development'or'refine'such'simulations'to'make'them'more'
accurate/informative.'
• explain'how'hypotheses'about'the'inheritance'of'a'trait'are'generated'and'how'they'are'revised'
based'on'additional'data.'
'
Pre6Lab*Assignment'(30'points)'
Read'the'article'and'be'prepared'to'discuss'it'in'lab.''You'should'write'down'one'thing'from'the'article'
that'was'interesting'(to'hand'in'at'the'beginning'of'classM1'paragraph)''
*
Introduction'
What'is'a'polygenic'trait?''What'is'an'environmental'effect?''What'is'a'GxE'interaction?''Be'sure'that'
you'understand'the'answers'to'these'three'questions'before'beginning'today’s'lab.'
'
In*Lab'
You'will'start'by'discussing'the'article.''After'discussing'the'article,'you'will'develop'a'simulation'of'
polygenic'inheritance'to'investigate'how'multiple'alleles'affect'the'risk'of'developing'a'particular'
disease.''You'will'also'examine'how'environment'affects'the'likelihood'of'developing'that'genetic'
disease.''Why'are'you'being'asked'to'develop'a'simulation'rather'than'simply'work'through'a'
simulation?''Working'through'simulations'is'very'valuable'if'you'are'paying'attention'to'detail'and'to'
what'the'simulation'reveals.''It'is'also'worth'developing'your'own'simulations'to'force'yourself'to'
carefully'think'through'the'details'of'a'process'and'check'that'you'are'simulating'it'accurately.''Such'a'
simulation'can'be'used'to'develop'a'model'for'a'process'or'for'the'inheritance'of'a'particular'condition.''
Models'are'valuable'in'biology:'they'can'be'used'to'generate'testable'hypotheses.''If'the'model'
correctly'depicts'the'inheritance'of'a'given'condition,'then'it'can'be'used'to'predict'what'will'happen'in'
various'cases.''If'the'prediction'is'correct,'this'provides'support'for'the'model.''If'the'prediction'is'
incorrect,'then'the'model'(and'the'hypothesis'that'the'model'reflects)'must'be'revised.''That’s'really'all'
there'is'to'mathematical'modeling'in'biology'–'you'develop'a'hypothesis,'create'a'model'that'is'based'
on'the'hypothesis,'generate'predictions'using'the'model,'and'test'those'predictions'against'data.''In'this'
way,'models'are'used'to'advance'our'understanding'of'both'basic'and'very'complex'biology.'''
'
In'today’s'lab,'you'will'develop'your'own'simulation'for'the'inheritance'of'heart'disease,'a'polygenic'
trait'that'is'affected'by'environmental'factors.''Are'you'going'to'have'instructions'for'how'to'structure'
your'simulation?''No!''That’s'the'point'of'today’s'exercise.''If'you'have'instructions'to'follow,'you’re'not'
making'your'own'simulation,'you’re'just'following'an'existing'simulation.''That’s'not'what'we’re'after'
today.'''
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Materials'
A'variety'of'materials'are'available'to'you.''You'can'also'feel'free'to'create'materials'of'your'own'or'ask'
for'additional'materials'if'necessary.'
• containers'
• paper'
• coins'
• +'and'M'alleles'for'
• markers'
• dice'
various'genes''
• lifestyle'cards'
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Directions:*Create*a*model*of*Polygenic*inhericance'
Work'in'groups'of'2M4.'
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Think'about'the'inheritance'of'heart'disease'(or'you'could'do'diabetes'or'some'other'disease).''Come'up'
with'a'hypothesis'for'the'number'and'effect'of'various'alleles'and'the'effect'of'environmental'factors.''
Create'a'simulation'that'illustrates'the'inheritance'and'expression'of'heart'disease.''Show'how'both'
genes'and'environment'affect'the'probability'of'an'individual’s'developing'the'disease.''Your'simulation'
should'include'the'following'aspects:'
• simulate'what'alleles'an'individual'gets'
• simulate'the'effect'of'the'environment'on'individuals'
• simulate'how'the'environmental'factor'interacts'differently'with'different'genotypes'
• simulate'children’s'genotypes'being'generated'from'parents''
• what'sorts'of'risks'children'inherit'from'their'parents'
'
If'you'get'stumped,'feel'free'to'talk'with'your'TA'or'with'other'groups'for'ideas.''You'may'not'simply'
take'another'group’s'idea,'but'you'may'get'input'from'them'to'help'structure'your'own'model.''Don’t'
get'frustrated!'''
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Explain*your*model*to*your*TA*(50*points).'Your'TA'will'either'ask'one'or'more'groups'to'present'their'
model'to'the'group,'or'ask'you'to'pair'up'with'another'group'to'explain'your'models'to'each'other.'
'
Type*up*your*model*(50*pts):'Once'your'model'works'the'way'that'you'want'it'to,'you'will'write'out'a'
short'(1M2'page)'description'of'your'simulation.'It'is'due'next'week'(please'type).''Be'sure'to'include'one'
paragraph'about'the'weaknesses'of'your'simulation'–'for'example,'where'it'may'be'oversimplified,'
where'it'may'not'be'quite'accurate,'etc.'
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Name:_______________*
*
*
Questions*(10pt*each)*Due*next*week*
Please*type*up*your*answers*and*print*them*(so*we*can*read*them)*
1.'If'a'parent'is'diagnosed'with'heart'disease,'does'that'mean'that'the'children'will'get'heart'disease?''
Explain.'''
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2.''Describe'at'least'3'ways'to'figure'out'if'you'have'a'high'risk'of'heart'disease.''
'
3.''When'you'think'about'genetic'screening'(like'at'23andme),'how'would'knowing'that'you'have'alleles'
associated'with'a'polygenic'disease'help'you'maintain'good'health?''How'could'it'have'a'negative'
impact'on'your'life'and'health?'
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3.'What'is'a'genotype'by'environment'interaction?'Why'is'it'important'for'a'trait'like'heart'disease?'
'
4.'When'scientists'say'they'found'a'gene'for'a'polygenic'trait'like'prostate'cancer'or'heart'disease,'what'
do'they'mean?'
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5.''Why'is'it'easier'to'map'Mendelian'traits'to'a'specific'region'of'the'genome'than'genes'associated'
with'polygenic'traits?'
6.''What'are'the'methods'by'which'the'location'of'a'gene'associated'with'a'particular'quantitative'trait'
can'be'narrowed'down?'
7.'Single'nucleotide'polymorphisms'(SNPs)'are'a'type'of'genetic'markers'can'be'useful'in'narrowing'the'
location'of'a'QTL.''Sketch/diagram'how'a'few'of'these'markers'would'be'arranged'on'a'chromosome'
and'use'your'illustration'to'explain'how'the'markers'are'used'to'find'QTLs.'(hand'sketch'is'fine)'
8.'How'does'DNA'sequencing'help'in'the'identification'of'QTLs?''What'can'DNA'sequencing'detect?'
9.'What'is'a'functional'test'of'a'candidate'gene?''Why'is'it'necessary.'
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Genetics Lab
23andMe
Name:________________________
23andMe (www.23andMe.com) is one of several companies offering personalized
SNP genotyping to the general public. You pay them $99, send them some spit (which
contains some of your cheek cells), and they genotype your DNA at hundreds of
thousands of SNP loci a DNA chip (also called a SNP chip). To learn about the DNA
chips (arrays) they use for SNP genotyping, check out this site:
https://www.23andme.com/more/genotyping/
Note: this genotyping technology uses DNA hybridization, not sequencing. It still costs
way more to get your whole genome sequenced.
Some of the SNPs genotyped by 23andMe are known to be associated with
genetic diseases. That is, individuals with one of the two possible alleles at the SNP
locus have an increased chance of getting the disease. This is either because the
genotyped SNP influences the chance of getting the disease or because a non-genotyped
SNP nearby in the genome (and in LD with the genotyped SNP) influences the chance of
getting the disease.
Go to the 23andMe web site (www.23andMe.com) and see how the genotypes of
the Mendel family tells them about their risk for various diseases. Select demo account,
and create an account. You can use a fake name and password if you prefer. When you
log in, you can learn about the results you would get if you purchased their service by
exploring the SNP results of the "Mendel Family," a real family, with their names
changed to protect their privacy. You can learn whether they have SNP alleles associated
with an increased chance of getting certain diseases, and learn how much their risk is
increased, and learn about their ancestry.
To view the Mendel family’s results, click on My Results at the top of the screen.
You can then choose what type of results you want to learn about (disease risk, drug
responses, ancestry, etc.)
Questions: (these are designed to guide you through a few of the 23andMe features)
1) (10 pts) Explore the site and check out the health traits or ancestry results of Greg
and Lilly Mendel. List something interesting you learned
2) (10 pts) Under “Health Overview/Health Risks” what are some diseases for which
Greg Mendel has an increased genetic risk?