Exercise: Mapping disease genes & Analyzing
microarray data
Objectives:
1. Develop an understanding of gene expression and the
means of identification and analysis.
- Review expression analysis, old and new
- Understand the basics of microarray analysis
- Appreciate different applications of microarrays
2. Understand polymorphisms.
- Compare types of polymorphisms and their origins
- Role of polymorphisms in health and disease
- Utility of polymorphisms in different types of
analysis
3. Understand the ethical issues related to
bioinformatics.
Introduction:
In Unit 2, you found how the use of genetic markers is
useful in comparative genomic mapping. Finding and using
genetic markers has long been recognized as being extremely
useful in a wide variety of applications. Examples include
recognition of disease gene polymorphisms, use in studies of
pathogens and epidemiology, selection of plants for
desirable agricultural characteristics such as seed yield or
height, and analysis of forensic evidence for civil and
criminal court cases. The combination of more means of
identification of polymorphisms along with the capability of
genomic analysis has brought considerable improvements in
these areas of inquiry. Additionally as a result of these
advances, other areas are undergoing rapid expansion and
development. Gene identification and association with
function(s) in metabolism, development and cell
differentiation, and various response systems are some
examples. Means of identifying polymorphisms include
restriction fragment length polymorphism [RFLP] and
use of probes for specific gene markers, either as part of
the gene in question or a closely linked marker to the gene
of interest.
Until recently, expression analysis has been accomplished
by studying one or a few genes at a time. With the advent of
microarrays, expression analysis has taken giant leaps
forward with the capability to screen in the range of 20,000
genes at a time. You will have the opportunity to become
familiar with the foundation of this powerful methodology in
preparation for the related project for this unit.
There are summary questions at the end of this
section. Points = 10. Due 10/9 midnight or
10/14.
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Pre-Exercise:
1. Review the following in any recent molecular
biology or genetics text.
- Gene mapping
- Genetic linkage
- Polymorphisms
- Acronyms to know
- EST
- RFLP
- SNP
- SSP
- STP
- VNTR
2. For an introduction to restriction
enzymes, try:
http://www.accessexcellence.org/AE/AEC/CC/restriction.html
http://www.ultranet.com/~jkimball/BiologyPages/R/RestrictionEnzymes.html
[This site also gives links to information on DNA
sequencing and recombinant DNA.]
3. For an online review of Hardy-Weinburg and for
the basics on human gene mapping, try Jacki Wicks and
T. P. Speed's lecture notes for Week 5: Genetic
epidemiology: Association and linkage:
http://oz.berkeley.edu/users/terry/Classes/s260.1998/Week5/week5/week5.html
4. For an introduction to the basics of
microarray methods, try the following animated
tutorial:
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
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Exercise:
Part 1: Exploring approaches to genetic
analysis
Tissue typing provides an excellent example of an
activity which has been accomplished using a variety of
methodologies over the years, including the development and
use of probes. Screening for human major histocompatibility
complex [MHC] antigens is required for tissue typing
of possible donors for organ transplants and of the
recipients. It is also useful in genetic linkage analysis
for a variety of disease associations. Originally, screening
was done using a cytotoxicity assay system for some of the
loci, and a mixed lymphocyte response [MLR] assay
for other loci. Use of RFLPs was introduced in the late
1980's followed by introduction of PCR methods in the
1990's. Microarrays are now being developed and introduced
for HLA screening.
To become familiar with HLA and tissue typing, try the
following tutorial which includes information on the
different screening methods:
http://www.aseatta.org.au/educatio.htm
For cutting edge approaches, the following papers are of
interest:
Feolo M, Fuller TC, Taylor M, Zone JJ, Neuhausen
SL, 2001. A strategy for high throughput HLA-DQ typing.
J Immunol Methods 2001 Dec 1;258(1-2):65-71
[Elsevier]
Cai H, White PS, Torney D, Deshpande A, Wang Z, Keller
RA, Marrone B, Nolan JP., 2000. Flow cytometry-based
minisequencing: a new platform for high-throughput
single-nucleotide polymorphism scoring. Genomics 2000 Jun
1;66(2):135-43. Erratum in: Genomics 2000 Nov 1;69(3):395
[Elsevier]
If you are interested in more resources on HLA and
typing, a worthy site to visit is the Anthony Nolan Bone
Marrow Trust:
http://www.anthonynolan.org.uk/HIG.
For a different application using probes, check out the
following site:
http://aem.asm.org/cgi/content/full/65/11/4775?view=full&pmid=10543785
The HLA allele with the strongest association with a
disease is HLA B27. It has a strong association frequency
with ankylosing spondylitis and one type of arthritis.
Therefore it is of interest to screen for the presence of
this particular allele. The complete mRNA sequence for B27
is in GenBank:
gi|187657|gb|M12678.1|HUMMHB27A
You may be interested in reading the following paper on
HLA-B27, which combines two computational methods to
identify peptide sequences from Chlamydia trachomatis
predicted to be involved in binding B27 and which may be
involved in the pathogenesis of B27 associated disease.
Kuon W, et al, 2001. Identification of
HLA-B27-restricted peptides from the Chlamydia
trachomatis proteome with possible relevance to
HLA-B27-associated diseases. J Immunol Oct
15;167(8):4738-4746.
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Part 2- Expression analysis using
microarrays
A. Explore microarrays. In the pre-exercise, you
should have had some fun with the animated tutorial. If not,
go back. For a more detailed exploration, try the
following:
http://www.bio.davidson.edu/courses/genomics/chip/chipreal.html
http://www.bsi.vt.edu/ralscher/gridit/intro_ma.htm
Resource list for microarrays:
http://www.deathstarinc.com/science/biology/chips.html
B. Design a microarray.
Using the original methods, about 165 allelic differences
were identified in the following loci of HLA: A, B, C, DR,
DQ, and DP. At the sequence level, the number of allelic
differences are far greater, and therefore an assay
detecting them would be more sensitive. Design a microarray
to rapidly screen for MHC antigens expressed on peripheral
blood WBCs.
1. Outline what you would need to make the arrays,
how you would design it to maximize data collection
[including all accessory controls], and how you
would intend to analyze the results.
2. To run the assay, let the following be
standards: positive controls are labeled red; unknowns
[samples] are labeled green. Give an example of some
expected results, including the resulting color of the
reactions.
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Summary Questions:
Try to limit your answers to 1-2 typed pages [12 pt
font]. This length should be sufficient for your
comments and any appropriate copy/pasted examples. [You
need not retype or copy/paste the questions as part of your
responses.]
1. Compare and contrast the following terms:
a. gene mapping vs. linkage analysis
b. RFLP vs. SNP analysis
2. Describe three approaches to analyzing disease
association with genetic markers.
3. Give two reasons why there can be genetic
marker-disease association but Hardy-Weinberg disequilibrium
at a marker locus.
4. Designing a microarray:
a. Outline what you would need to make
the arrays, how you would design it to maximize data
collection [including all accessory controls],
and how you would intend to analyze the results.
b. To run the assay, let the following be
standards: positive controls are labeled red; unknowns
[samples] are labeled green. Give an example of
some expected results, including the resulting color of
the reactions.
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