Post on 02-Jun-2018
8/10/2019 SCD Presentation
1/23
HBBGene Analysis in Determining Sickle Cell Disease
Rebecca Plessel and Emily Cribas
BIOL 230M
December 5, 2014
1 / 1 4
8/10/2019 SCD Presentation
2/23
Introduction
The HBB Gene
Codes for globin, a protein that creates half of the hemoglobin inred blood cells1
Is located on:
Cytogenic location: 11p 15.5
Figure 1: Gene location
1HBB Gene. Genetics Home Reference. National Library of Medicine, 24 Nov. 2014.
Web. 25 Nov. 2014.2 / 1 4
8/10/2019 SCD Presentation
3/23
Introduction
The HBB Gene
Molecular location on chromosome 11: bp 5,222,465 - 5,227,0701
72,20072,10072 K71,90071,80071,70071,60071,50071,40071,30071,20071,10071 K70,90070,80070,70070,60070,50070,40070,30070,200
Sequence
HBBNM_000518.4 NP_000509.1
exon 1 exon 2 exon 3
Genes
NM_0005... NM_000518.4 NM_000518.4
BLAST Results for: ref|NM_000518.4| (626 letters)
NM_000518.4
Cleaned Alignments - BLAST Results for: ref|NM_000518.4| (626 letters)
Figure 2: GenBank Sequence
1HBB Gene. Genetics Home Reference. National Library of Medicine, 24 Nov. 2014.
Web. 25 Nov. 2014.3 / 1 4
8/10/2019 SCD Presentation
4/23
Introduction
Common HBBMutations
The most common mutations are due to single point mutations1:
-thalassemia
1Rees, D. C. et al. LancetDec. 2010, 376, 201831.4 / 1 4
8/10/2019 SCD Presentation
5/23
Introduction
Common HBBMutations
The most common mutations are due to single point mutations1:
-thalassemia
Methemoglobinemia
1Rees, D. C. et al. LancetDec. 2010, 376, 201831.4 / 1 4
8/10/2019 SCD Presentation
6/23
Introduction
Common HBBMutations
The most common mutations are due to single point mutations1:
-thalassemia
MethemoglobinemiaHemoglobin C
1Rees, D. C. et al. LancetDec. 2010, 376, 201831.4 / 1 4
I d i
8/10/2019 SCD Presentation
7/23
Introduction
Common HBBMutations
The most common mutations are due to single point mutations1:
-thalassemia
MethemoglobinemiaHemoglobin C
Hemoglobin E
1Rees, D. C. et al. LancetDec. 2010, 376, 201831.4 / 1 4
I t d ti
8/10/2019 SCD Presentation
8/23
Introduction
Common HBBMutations
The most common mutations are due to single point mutations1:
-thalassemia
MethemoglobinemiaHemoglobin C
Hemoglobin E
Sickle Cell Anemia
1Rees, D. C. et al. LancetDec. 2010, 376, 201831.4 / 1 4
Introduction
8/10/2019 SCD Presentation
9/23
Introduction
Sickle Cell Anemia (HbS)
Codon GAA/GAG (glutamic acid) chages to GUA/GUG (valine),
causing deformation2
Figure 3: Point Mutation
Common problems include:
Fatigue
Organ damage
Extreme pain
Blocked blood vessels
Frequent infection
High blood pressure
Heart failure
2Rees, D. C. et al. LancetDec. 2010, 376, 201831. 5 / 1 4
Introduction
8/10/2019 SCD Presentation
10/23
Introduction
SCD Treatment
hiPSCs3
Human induced pluripotent stem cellsSome have problems differentiating
Not very reliable
Hydroxyurea4
Increased HbF levelsCarcinogenicWorks well or not at all
Bone Marrow Transplant
Risky85% cured
More tests for better drugs3Sun, N.; Zhao, H. Biotechnology and bioengineeringMay 2014, 111, 104853.
4Akinsheye, I. et al. en BloodJuly 2011, 118, 1927.6 / 1 4
Methods
8/10/2019 SCD Presentation
11/23
Methods
Methods
1 DNA Extraction
Buccal Swab Protocol
2 DNA Isolation and HVI PCR Amplification
3 Gel Electrophoresis
If bands, alter master mix to get stronger bands
If no bands, either re-run PCR or alter master mix
7 / 1 4
Results
8/10/2019 SCD Presentation
12/23
Results
Different Master Mix Concentrations
Table 1: Original Master Mix
Item
Ingredient Concentration (l)
dNTP 2.510x Buffer 2.5Left Primer 0.5Right Primer 0.5
Taq Polymerase 0.5DNA 2.0dH2O 16.5
Table 2: Altered Master Mix
Item
Ingredient Concentration (l)
dNTP 2.510x Buffer 2.5Left Primer 1.0Right Primer 1.0
Taq Polymerase 0.5DNA 2.0dH2O 15.5
*Note: DNA was added on a per tube basis
8 / 1 4
Results
8/10/2019 SCD Presentation
13/23
First Set of Primers
Figure 4: Left Primer
Figure 5: Right Primer9 / 1 4
8/10/2019 SCD Presentation
14/23
Results
8/10/2019 SCD Presentation
15/23
Gel Electrophoresis 1 & 2
Figure 7: DNA with OriginalConcentrations of Master Mix, 25 l
Figure 8: DNA with SameConcentration, 50 l
11/14
Results
8/10/2019 SCD Presentation
16/23
Gel Electrophoresis 3 & 4
Figure 9: DNA with Different PrimerConcentrations (0.5 and 1.0 l) withOriginal DNA Sample
Figure 10: Stock Solution with 0.5 and1.0 l Concentration of ReorderedPrimers
12/14
Discussion
8/10/2019 SCD Presentation
17/23
Discussion
DNA with primer only worked with the first 2 tries (7,8)
13/14
Discussion
8/10/2019 SCD Presentation
18/23
Discussion
DNA with primer only worked with the first 2 tries (7,8)
Changing concentrations of primer did not work (9)
13/14
8/10/2019 SCD Presentation
19/23
Discussion
8/10/2019 SCD Presentation
20/23
Discussion
DNA with primer only worked with the first 2 tries (7,8)
Changing concentrations of primer did not work (9)
Using stock solution with these primers did not work as well (10)
This led us to believe the problem were the primers
13/14
Discussion
8/10/2019 SCD Presentation
21/23
Discussion
Running the PCR with reordered primers did not work either (10)
14/14
Discussion
8/10/2019 SCD Presentation
22/23
Discussion
Running the PCR with reordered primers did not work either (10)
Most likely reasons PCR/Gel didnt amplify/exhibit bands:
Primer degradation by multiple freeze-thaw cycles
Formation of primer dimers
Using cheek swab DNA vs DNA from blood
Protease may not have broken down all protein in DNA (i.e. histones)
14/14
Discussion
8/10/2019 SCD Presentation
23/23
Discussion
Running the PCR with reordered primers did not work either (10)
Most likely reasons PCR/Gel didnt amplify/exhibit bands:
Primer degradation by multiple freeze-thaw cycles
Formation of primer dimers
Using cheek swab DNA vs DNA from blood
Protease may not have broken down all protein in DNA (i.e. histones)
PCR is finicky and would ideally be performed more than once a week
14/14