Transcript 2011_InstructorSlidesR

DNA Sequencing Research Projects for Summer
Programs or Undergraduate Courses
Julie A. Emerson
Department of Biology
Amherst College
Amherst, MA 01002
Objectives of Group Research Projects
• Enable participants to use the polymerase chain reaction
(PCR) and DNA sequence analyses to discover something new
about themselves or the surrounding microbial environment
• Run 2-3 different projects, to keep group number to a
manageable size and so different groups can present to and
learn from each other
• Select genes for study that have easily-identifiable differences
in DNA sequence in the test population, so that comparisons
can be made between test subjects
• For projects using human subjects, select genes that are
associated with interesting human traits
• Avoid selecting genes that are associated with human
diseases/disorders or that may raise questions about paternity
• The time frame of the projects, from introduction to final
presentations, must fit within a 10-12 day window for summer
program, or in several 2-4 hour lab periods for a course
Four projects have been designed and
implemented since 2005:
Project #1 – Molecular Identification of Human Polymorphisms in
the Melanocyte-Stimulating Hormone Receptor (MC1R) Gene
Project #2 – Adult Lactase Persistence and Associated
Polymorphisms in the Human Lactase Phlorizin Hydrolase Gene
Project #3 – Identification of Single Nucleotide Polymorphisms in a
Human Bitter Taste Receptor Gene
Project #4 – Molecular Identification of Bacteria in the Environment
Group Research Projects #1-3 : Molecular Identification of Human
Single Nucleotide Polymorphisms
Day 1:
Volunteers fill out Human Subjects Questionnaire
↓
DNA isolation from human cheek cells
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PCR using MC1R, lactase or PTC forward and reverse primers
↓
Day 2:
Analytical agarose gel of PCR products
↓
Day 3:
Spin purification of PCR products
↓
Analytical agarose gel of purified PCR products and
quantification of DNA amounts
↓
Sequencing reactions set up and shipped to
sequencing center via overnight delivery
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Day 4:
DNA sequencing by BRC at Cornell University
↓
Days 5 & 6:
DNA sequence analysis and
presentation preparation
↓
Day 7:
Project Presentation
Group Project #4 - Molecular Identification of Bacteria in the Environment
Day 1:
Day 2:
Day 3:
Day 4:
Days 5 & 6:
Day 7:
Inoculation of nutrient agar plates
↓
Incubate overnight at 37ºC
↓
Secondary streaking for methylene blue staining and
DNA isolation from single bacterial colonies using
PrepMan™ Ultra Sample Preparation Reagent
↓
PCR using MicroSeq® 500 16S rDNA PCR kit
↓
Analytical agarose gel of PCR products
↓
Spin purification of PCR products
↓
Analytical agarose gel of purified PCR products and
quantification of DNA amounts
↓
Sequencing reactions set up and shipped to
sequencing center via overnight delivery
↓
DNA sequencing by BRC at Cornell University
↓
Methylene blue staining of isolates,
DNA sequence analysis and presentation preparation
↓
Project Presentation
Steps in
green same
as in human
projects
Let’s do part of the human bitter
taste receptor project right now!
A. Phenotyping with phenylthiocarbamide (PTC)
taster strips
B. Background on the molecular genetics of the
PTC taste receptor
C. Analyze DNA sequence that “matches” your
phenotype - can you find the polymorphisms?
D. Location of the amino acid polymorphisms within
the 3-D structure of the PTC taste receptor
A. What is your PTC taster phenotype?
1. Put paper strip flat on surface of your tongue
for a few seconds
2. Repeat with second strip
3. Any difference between the two?
4. Rearrange yourselves into groups based on
PTC sensitivity:
- strong bitter taste
- medium bitter taste
- weak bitter taste
- no bitter taste
B. Phenylthiocarbamide (PTC) taste receptor
• The inability to taste certain compounds is usually
due to simple, recessive Mendelian inheritance.
• Dozens of taste and odorant receptors have been
cloned and sequenced in the last 20 years.
• The TAS2R28 gene encodes a bitter taste receptor
that enables humans to taste the compound PTC.
• The PTC (TAS2R28) gene has a single coding
exon, for a polypeptide chain with 333 amino acids.
B. PTC taste receptor, continued
• Three common single nucleotide polymorphisms
(SNPs) are associated with PTC sensitivity.
• Each SNP results in a change to the amino acid
sequence of the PTC receptor.
Table 1. Polymorphisms within the PTC gene
Position
Position
SNP
Amino Acid
(bp)
(amino acid) Allele
Encoded
145
49
C or G
Pro or Ala
785
262
C or T
Ala or Val
886
296
G or A
Val or Ile
• The SNPs are usually inherited together in certain
combinations, e.g., haplotypes.
Table 2. SNP haplotypes of the PTC gene within two study groups
(named for the first letter of the amino acid present at positions 49, 262 and 296)
Haplotype
European Freq. East Asian Freq.
PAV
49%
70%
AVI
47%
30%
AAV (from recomb. at aa 49)
3%
-
A later screen identified two additional haplotypes, PVI and AAI, which were
found only in individuals of sub-Saharan African ancestry. The AVI haplotype was
found in all populations except Southwest Native Americans (Kim et al., 2003).
• Certain haplotypes are generally correlated with taster status.
Table 3. Genotype association with taste phenotypes (by haplotypes)
Genotype (diploid)
AVI/AVI (73)
AVI/AAV (21)
*/PAV (170)
Nontasters
81%
52%
2%
Tasters
19%
48%
98%
*= PAV, AVI or AAV. The total number of PTC genotypes observed was 5, as
no AAV homozygotes were observed in the study group (Kim et al, 2003).
C. Your goal:
Examine the DNA sequence chromotograms, locate the areas
of the three SNPs and identify ‘your’ amino acid haplotype.
Note: remember you are a diploid organism!
C. DNA Sequence Analysis of the PTC gene
1. Use sequence handout and Table 1 to determine ‘your’
DNA sequence at the position of each of the three SNPs
2. What is your diploid amino acid haplotype?
3. Does this haplotype correspond with PTC taster status?
4. Any instances of heterozygosity?
bp 145
aa #49
C. 4. What about heterozygosity?
bp 785
a.a. #262
Note the overlapping blue (C) and red (T) peaks.
(The pink N above – inserted when the sequencing computer cannot make a clear
call - is a strong hint that something is up at that position!)
C. 4. What about heterozygosity, cont.?
What are the possible diploid
amino acid haplotypes for this
individual?
Answer:
PAV/AVI (most likely)
But cannot rule out:
PAI/AVV
PVV/AAI
PVI/AAV
(due to recombinant chromosomes
in the population)
D. Why are people with the AVI/AVI genotype
unable to taste PTC?
1. What is the normal structure and function of the
PTC receptor?
2. Where within the structure do the non-taster
variant amino acids reside?
3. What is the side chain structure of the taster
(PAV) and non-taster amino acids (AVI)?
4. How do the variant amino acids alter the structure
and/or function of the protein?
1. The PTC receptor is a G-protein linked receptor, with
seven transmembrane domains.
A taster PTC receptor will bind PTC with its extracellular
domain and signal by activating a heterotrimeric G protein
on the cytoplasmic surface of the membrane.
Pathways of Bitter and Sweet Taste Transduction
Margolskee, R. et al., J Biol Chem. 277:1-4, 2002
A non-taster PTC receptor (green above) may either
a) not bind PTC
b) bind PTC but be defective in activating the heterotrimeric G protein
Ligand binding studies indicate that taster and non-taster
receptors have the same binding affinity for PTC.
Sum: Extracellular domain of receptor likely unaffected in non-tasters.
Predicted 3D Structures for PTC Bound to Bitter Receptor
Floriano et al. J Mol Model, (2006) 12: 931-941
Taster Variant
Non-Taster Variant
2. Location of the Variant Amino Acids in
PTC Tasters (PAV) and Non-Tasters (AVI)
PTC Tasters (PAV)
Non-Tasters (AVI)
Amino acid (aa) 49 is located at cytoplasmic base of the
first transmembrane region and aa 262 and 296 reside
within transmembrane regions #6 and #7, respectively.
3. Amino Acid
Structures
49: Pro -> Ala
262: Ala -> Val
296: Val -> Iso
4. Predicted 3D Structure for the PTC Receptor
Unbound
Bound (note shift in TM6)
TM6 Position 262 and TM7 Position 296
Unbound
Bound (note change in TM6)
For AVI (non-taster) haplotype, V at 262 and I at 296 prevent
interaction between transmembrane regions 6 and 7
‘VI’ mutant variations shown as gray shadows
Floriano et al. J Mol Model, (2006) 12: 931-941
D. Conclusions From The Models
• The extracellular PTC binding sites between taster
and non-taster receptors are not significantly different
• Non-tasting in AVI haplotype is likely due to V-262 and
I-296; aa 49 has minimal impact
• Consistent with PVI = non-taster
• AA sites 262 and 296 have the potential to interfere
with TM6 and TM7 interaction (V and I are bulkier than
A and V, respectively)
• It is hypothesized that movement of TM6 is necessary
for activation of the heterotrimeric G protein
More Fun Informaton about PTC Receptor
• PTC taste sensitivity displays a broad and continuous
distribution (e.g., it behaves like a quantitative trait).
• On average, PTC taste sensitivity is highest for the
PAV/PAV (taster) homozygotes, slightly but significantly
lower for the PAV heterozygotes, and lowest by far for the
AVI/AVI (non-taster) homozygotes.
• More rare AVI/AAV heterozygotes have a mean PTC score
slightly, but significantly, higher than the AVI/AVI
homozygotes.
• All non-human primates examined to date are homozygous
for the PAV (taster) haplotype. Thus, the AVI nontaster
haplotype arose after humans diverged from the most
recent common primate ancestor.
• There are non-taster chimps: same gene, but different
mutation than humans => molecular convergent evolution!!