Teaching Biology Through
Bioinformatics
Real world genomics research
in your classroom
Kim B. Foglia
Stuart M. Brown, PhD
Division Ave. High School
AP Biology
Levittown
Director of Research Computing
2004-2005
NYU School of Medicine
Bioinformatics
 Use of computers to analyze genomic
data for molecular biology research
basic biological research
 medical research
 clinical medicine application
 taxonomy

Bioinformatics
2005-2006
Bioinformatics is a great way
to learn biology
 Using computers to study primary
biological data (genomes, proteins,
other databases)
students learn biology as a dynamic
process of interpreting complex data
 students can reproduce current scientific
work & ask new questions

 science is no longer just a collection of facts
in a textbook, it’s a process of inquiry
Bioinformatics
2005-2006
Teaching Biology with Bioinformatics
 Bioinformatics tools lead directly to
insights about genes, proteins, evolution
“Hey! Most human genes have more
bases of introns than they do exons.”
 “Hemoglobin sequences show that seals
are closer to weasels than they are to
whales.”
 “Protein shapes determine their function,
so small changes can make a big
difference.”

Bioinformatics
2005-2006
All you need…
 Students can work on
bioinformatics questions at low cost
only need Internet connected computers
 most database tools are free on Internet

 unlimited data
 GenBank, protein structures, mutations,
microarrays, etc.
teacher knowledge & “comfort”
 questions to answer

Bioinformatics
2005-2006
What we offer…
 Teaching modules

inquiry labs
 student & teacher lab handouts

supporting teaching resources
 PowerPoint presentations
 Supplemental skills modules
 Download from Web site

http://bio.kimunity.com
Bioinformatics
2005-2006
Modules in development
 Testing for Sickle Cell Anemia

develop a genetic diagnostic test to
screen for disease
 Endosymbiosis

evolutionary history mitochondria &
chloroplasts
 Hemophilia Gene Therapy

build a vector and insert correct gene
 Are Seals and Whales Related?

studying evolutionary relationships
Bioinformatics
2005-2006
Using Bioinformatics
in Medicine
Sickle Cell Anemia &
the Hemoglobin Gene
AP Biology
2004-2005
Sickle Cell Anemia
 Most common genetic disease in US



high incidence in African-Americans
affects red blood cell structure & function
single base mutation causing single amino acid change
 SNP = single nucleotide polymorphism
Bioinformatics
2005-2006
Symptoms
 Anemia

jaundice, fatigue, paleness, shortness of breath
 Hypoxia (low oxygen) & capillary damage


severe pain in organs & joints
retinal damage (blindness)
 Delayed growth

delayed puberty, stunted growth
 Infections



more susceptible
depressed immune
death from bacterial infections
 Stroke


blocked small blood vessels in brain
primarily in children
Bioinformatics
2005-2006
Sickle cell hemoglobin
Bioinformatics
mutant hemoglobin (Hb S)
2005-2006
Bioinformatics
2005-2006
Cell biology
 Hb S molecules stick
together
form fibers
 under low blood
oxygen levels

 deoxyhemoglobin
sickles

distortion of cells
from normal round to
sickle shape
Bioinformatics
2005-2006
Genetics
 Sickle cell mutation



Hb S
changes 6th amino acid of  hemoglobin chain
normal glutamic acid  valine
 Recessive allele

heterozygote
 Hb AS, normal, but carrier

Hb A
Hb S
2 sickle cell carriers mate… Hb A HbAA
HbAS
homozygote recessive
 Hb SS, sickle cell disease

 each child has 1/4 chance
of having the disease
Hb S HbAS
Bioinformatics
HbSS
2005-2006
Prevalence in U.S.
 Carriers
~2 million Americans carry sickle cell
trait
 1 in 14 African-Americans

 Disease
~72,000 Americans have disease
 ~1 in every 700 African-American babies
born in U.S. has sickle cell disease

Bioinformatics
2005-2006
The Malaria Connection
 Sickle cell disease is surprisingly common
for a potentially lethal genetic disease
 Heterozygote advantage

heterozygotes are tolerant of malaria
infection & do not suffer symptoms of sickle
cell disease
Bioinformatics
2005-2006
Malaria
Bioinformatics
2005-2006
Prevalence of Malaria
Prevalence of Sickle
Cell Anemia
~sickle cell movie~
Bioinformatics
2005-2006
Public health
 Many carriers of this mutant allele are
not aware that they have it

at risk of having children with the
disease
 DNA test for sickle cell allele would
benefit public health
genetic counseling
 pre-natal testing

Bioinformatics
2005-2006
Your Assignment
 Develop a simple inexpensive DNA
test for sickle cell allele
 develop DNA probe
 test for presence of sickle cell
mutation

use bioinformatics tools
 online databases of DNA sequences
 UCSC Genome Browser
 probe design tool
 Primer3
Bioinformatics
2005-2006
DNA review
 DNA double helix
A–T, C–G
 base pair bonds can be broken
by heating to 100°C

 separate strands
 denature, or melt
Bioinformatics
2005-2006
DNA probes
 Probe


short, single stranded DNA molecule
mix with denatured DNA
 DNA Hybridization

probe bonds to complementary DNA sequence
 Label

probe is labeled for easy detection
labeled probe
genomic DNA
Bioinformatics
3’
G A T C A G T A G
C T A G T C A T C
2005-2006
5’
Designing Probes
 Allele specific probes
probes require matched sequences
 can detect single base differences in
alleles
 single mis-matched base near middle of
probe greatly reduces hybridization
efficiency

labeled probe
genomic DNA
X
C T A G T C A T C
3’
Bioinformatics
5’
2005-2006
Dot blot
 Genomic DNA
denature DNA
 bind DNA from cells on filter paper

 DNA hybridization
wash probe over filter paper
 if complementary sequence present,
probe binds to genomic DNA
 expose on X-ray film

 dark spots show bound probe
Bioinformatics
2005-2006
Get hemoglobin sequence
 UCSC Genome Browser
human genome database
 http://genome.ucsc.edu/






Bioinformatics
UCSC Genome Browser home page
click on link to Genome Browser
in genome pulldown menu, choose “Human”
for position text box, type “HBB” (hemoglobin )
hit “submit”
2005-2006
Genome Browser Results
 Listing of genes & sequences in
database

Click on “RefSeq” gene for HBB (NM_000518)
Bioinformatics
2005-2006
Chromosome view
 Position of HBB in genome

at base 5.2 million on chromosome 11
Bioinformatics
2005-2006
Change view of chromosome
 Move & zoom tools

zoom out ~30x to see more of
chromosome 11
Bioinformatics
2005-2006
More Hb genes
 Cluster of hemoglobin genes on
chromosome 11
HBD, HBG1, HBG2 & HBE1
 what are these genes?

Bioinformatics
2005-2006
Get the DNA sequence
 Click on the HBB RefSeq gene

HBB RefSeq summary page
Bioinformatics
2005-2006
HBB RefSeq gene summary page
 Click on “Genomic Sequence from
assembly”
Bioinformatics
2005-2006
Formatting the sequence
 Sequence Formatting Options
“exons in upper case, everything else in
lower case”
 hit “submit”

 Genomic DNA

lower case = introns
 spliced out of mRNA before translation

upper case = exons
 translated into polypeptide chain
Bioinformatics
2005-2006
HBB DNA sequence
>hg16_refGene_NM_000518 range=chr11:5211005-5212610 5'pad=0 3'pad=0 revComp=TRUE
ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC
ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGG
CAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGgttggtat
caaggttacaagacaggtttaaggagaccaatagaaactgggcatgtgga
gacagagaagactcttgggtttctgataggcactgactctctctgcctat
tggtctattttcccacccttagGCTGCTGGTGGTCTACCCTTGGACCCAG
AGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGG
CAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTG
ATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGT
GAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGgtgag
tctatgggacgcttgatgttttctttccccttcttttctatggttaagtt
catgtcataggaaggggataagtaacagggtacagtttagaatgggaaac
agacgaatgattgcatcagtgtggaagtctcaggatcgttttagtttctt

ttatttgctgttcataacaattgttttcttttgtttaattcttgctttct
ttttttttcttctccgcaatttttactattatacttaatgccttaacatt
gtgtataacaaaaggaaatatctctgagatacattaagtaacttaaaaaa
aaactttacacagtctgcctagtacattactatttggaatatatgtgtgc
ttatttgcatattcataatctccctactttattttcttttatttttaatt
gatacataatcattatacatatttatgggttaaagtgtaatgttttaata
tgtgtacacatattgaccaaatcagggtaattttgcatttgtaattttaa
aaaatgctttcttcttttaatatacttttttgtttatcttatttctaata
ctttccctaatctctttctttcagggcaataatgatacaatgtatcatgc
ctctttgcaccattctaaagaataacagtgataatttctgggttaaggca
Bioinformatics
2005-2006
atagcaatatctctgcatataaatatttctgcatataaattgtaactgat
 first 50 bases are
untranslated “leader”
sequence
 actual protein coding
sequence starts at
base 51
starting with
letters ATG
Get the mutant sequence
 Sickle cell mutation



single base mutation
6th amino acid: glutamic acid  valine
need DNA sequence to design probe
 SNPs


single nucleotide polymorphisms
“variations and repeats” section: pack
Bioinformatics
2005-2006
SNPs of HBB gene
 several SNPs of HBB gene
need mutation in exon
 near beginning of HBB protein
 rs334 = Hb S mutation

Bioinformatics
2005-2006
rs334 Hb S sickle cell mutation
 “Sequence in Assembly” = normal sequence
 “Alternate Sequence” = sickle cell sequence
Bioinformatics
2005-2006
Align Hb A & Hb S sequences
 Line up sequences
Normal:
HBB:
Mutant:

catggtgcacctgactcctgAggagaagtctgccgttactg
ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGG
catggtgcacctgactcctgTggagaagtctgccgttactg
sequence fragment is enough to design
DNA probes for normal & mutant
sequences
Bioinformatics
2005-2006
Designing the probe
 Primer3

free on Web from MIT
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

powerful tool for primer design
 paste in sequence fragment
Bioinformatics
2005-2006
Allele specific probes
 Need 2 probes
normal allele probe
 sickle cell allele probe
 choose hybridization probes

 Customize probes
12-16 bases
 40°-60°C

longer probes are stable
at higher temperatures
Bioinformatics
2005-2006
Your probes…
 Ready to order!
 Place an order at your local DNA lab!
Bioinformatics
2005-2006
Extra credit
Advanced Assignments
AP Biology
2004-2005
Advanced Assignment #1
 Use the Web to research other “allele
specific” genotyping methods
ligase chain reaction
 primer extension
 TaqMan

 Design probes for one of these
alternate technologies
Bioinformatics
2005-2006
Advanced Assignment #2
 PCR & Restriction Digest

pre-natal testing
 for small samples it is necessary to use
PCR to amplify the amount of genomic DNA
before testing
 once you have a PCR-amplified DNA
fragment of a gene, a restriction enzyme
may be able to distinguish between alleles

design PCR primers & find restriction
enzyme that will locate sickle cell allele
 design with Primer3
Bioinformatics
2005-2006
Restriction enzymes
 NEBcutter
http://tools.neb.com/NEBcutter2
 New England BioLabs
 screens DNA sequence against all
restriction enzymes

 Webcutter
similar program
 http://www.firstmarket.com/cutter/cut2.html

Bioinformatics
2005-2006
NEBcutter
Bioinformatics
2005-2006
Advanced Assignment #3
 Population genetics

determine if sickle cell allele is in HardyWeinberg equilibrium in the U.S.
African-American population
 ~2 million Americans carry sickle cell trait
 1 in 14 African-Americans is a carrier
 ~1 in every 700 African-American babies
born in U.S. has sickle cell disease
Bioinformatics
2005-2006