AP & Regents Biology
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Transcript AP & Regents Biology
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
Bioinformatics
3’
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