Download Hemoglobin Beta

Hemoglobin Beta-subunit (HBB)
Josh Bram and Wilton Smith
What is Hemoglobin?
• Oxygen (and CO2) transporter
for all vertebrates
• Tetramer composed of four
protein subunits (two alpha
and two beta subunits)
• Four Iron-containing haeme
centers carry one oxygen
molecule each
• Beta-subunit mutations cause:
– Sickle-cell anemia
– Beta-thalessemia
• 146 amino acid protein
subunit
Hemoglobin - β Mutation Diseases
Sickle Cell Anemia
• Mutation causing betaglobin units to stick
together in masses and
deform Red Blood Cells
• RBCs take crescent shape
and have many
consequences
• These include premature
death and blocking of small
blood vessels
Beta Thalassemia
• Either a decrease or
absence of beta-globin
production
• Prevents Red Blood Cells
from having enough
Hemoglobin to supply
oxygen to the body
• In mice, leads to sickly and
weak individuals, or
stillborn offspring
Peromyscus leucopus
Primer Design
• Targeted Intron 2 (~700 base pairs)
• Forward Primer (E2E3F):
5’ ctccatgtggatcctgagaac 3’
– GC%: 52.38
– 21 base pairs
– Melting point: 55° C
• Reverse Primer (E2E3R):
5’ acgatcatattgcccaggag 3’
– GC%: 50.00
– 20 base pairs
– Melting Point: 55° C
DNA Extraction
• Qiagen DNeasy Blood & Tissue Kit
• Two samples of P. leucopus extracted
– Sample 1 extracted after 20 minute incubation
– Sample 2 extracted after extended incubation
• Both DNA samples yielded amplification
results at one time or another; however,
sample 2 proved to be a more reliable DNA
source
Polymerase Chain Reaction (PCR)
• 6.25 μL Mastermix
– Mix composed of parts:
• 50 x 1.25 μL 10x buffer = 62.5 μL
• 50 x 1.25 μL dNTP = 62.5 μL
• 50 x 0.1 μL Taq Polymerase = 5 μL
•
•
•
•
4.25 μL dH2O
1.00 μL DNA
0.50 μL PF
0.50 μL PR
Initial Identification
• Six sample PCR
– Sample DNA 1 at 50˚C,
55˚C, and 60˚C
– Sample DNA 2 at 50˚C,
55˚C, and 60˚C
• Successful gene
amplification for
Sample DNA 2 at 60˚C
PCR (well seven)
2 kb
1.5 kb
1 kb
700 bp
500 bp
300 bp
100 bp
What happened?
• Only Sample DNA 2
used for future PCR
reactions
• Gene amplification
failed to occur at 60˚C
(or any temperature)
• Possible errors in PCR
mix setup, PCR machine
setup, random error,
denatured primers
Successfully Identified
• PCR run at multiple
primer concentrations
(1X, 1.5X, 2X) for
Sample DNA 2
• Successful gene
amplification for across
all primer
concentrations (wells
two, three, and four)
1, 1.5, and 2x primer concentrations all
show amplification at approx. 700 bp
Initial 50 μL PCR products
• 50 μL PCR reactions for
eventual sequencing
• All PCR ingredient
volumes multiplied by
four for 50 μL PCR
• Wells two and three
show the HBB PCR
products (1X and 1.5X
Primer concentrations)
HBB PCR Prodcuts, approx. 700 bp
PCR Product Cleanup for Analysis
• Used Qiagen PCR Purification Kit
• Cleanup up PCR Product to send for
sequencing
• Sent 5 μL of each primer, 10 μL of PCR product
• Sent to Penn State sequencing facility in
Chandlee building
HBB Sequence
HBB Forward Primer
560
580
600
620
640
660
680
700
720
740
760
780
800
820
840
860
880
900
920
940
960
Sequence
BLAST Results for: Nucleotide Sequence (642 letters)
227405
227405
227405
• Compared to
Mus sequence
980
1K
1,020
1,040
HBB Reverse Primer
460
480
500
520
540
560
580
600
620
640
660
680
700
720
740
760
780
800
820
840
860
880
900
920
940
960
Sequence
BLAST Results for: Nucleotide Sequence (612 letters)
118707
• Compared to
Mus sequence
118707
980
1K
1,020
1,040
BLAST Results
Population Samples
• Six Population Sample
PCR at 60˚C
–
–
–
–
–
–
TK20806
TK20808
TK20809
TK20813
TK20852
TK20857
East
West
• All samples showed
gene amplification
5E (806) vs 9W (852)
Conserved vs Unconserved
Site 179
Depending on reading frame: Cys to Tyr, Val to Met, Leu to Leu
Bibliography
• http://www.uniprot.org/uniprot/P68871
• http://ghr.nlm.nih.gov/gene/HBB
• http://biotools.umassmed.edu/bioapps/primer3_
www.cgi
• http://www.ncbi.nlm.nih.gov/genbank/
• http://www.megasoftware.net/
• http://nucleobytes.com/index.php/4peaks
• http://bioinformatics.picr.man.ac.uk/research/sof
tware/tools/sequenceconverter.html