HBB Gene Analysis in Determining Sickle Cell Disease

Rebecca Plessel and Emily Cribas

BIOL 230M

December 5, 2014

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Introduction

The HBB Gene

Codes for globin, a protein that creates half of the hemoglobin in
red blood cells1
Is located on:
Cytogenic location: 11p 15.5

Figure 1: Gene location

HBB Gene. Genetics Home Reference. National Library of Medicine, 24 Nov. 2014.
Web. 25 Nov. 2014.
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Introduction

The HBB Gene

Molecular location on chromosome 11: bp 5,222,465 - 5,227,0701

70,200

70,300

70,400

70,500

70,600

70,700

70,800

70,900

71 K

71,100 71,200

71,300

71,400

71,500 71,600

71,700

71,800

71,900 72 K

72,100 72,200

Sequence
Genes
HBB
NM_000518.4
exon 1

NP_000509.1
exon 2

exon 3

BLAST Results for: ref|NM_000518.4| (626 letters)

NM_0005...

NM_000518.4

NM_000518.4

Cleaned Alignments - BLAST Results for: ref|NM_000518.4| (626 letters)

NM_000518.4

Figure 2: GenBank Sequence

HBB Gene. Genetics Home Reference. National Library of Medicine, 24 Nov. 2014.
Web. 25 Nov. 2014.
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Introduction

Common HBB Mutations

The most common mutations are due to single point mutations1 :

-thalassemia

Rees, D. C. et al. Lancet Dec. 2010, 376, 201831.

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Introduction

Common HBB Mutations

The most common mutations are due to single point mutations1 :

-thalassemia
Methemoglobinemia

Rees, D. C. et al. Lancet Dec. 2010, 376, 201831.

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Introduction

Common HBB Mutations

The most common mutations are due to single point mutations1 :

-thalassemia
Methemoglobinemia
Hemoglobin C

Rees, D. C. et al. Lancet Dec. 2010, 376, 201831.

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Introduction

Common HBB Mutations

The most common mutations are due to single point mutations1 :

-thalassemia
Methemoglobinemia
Hemoglobin C
Hemoglobin E

Rees, D. C. et al. Lancet Dec. 2010, 376, 201831.

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Introduction

Common HBB Mutations

The most common mutations are due to single point mutations1 :

-thalassemia
Methemoglobinemia
Hemoglobin C
Hemoglobin E
Sickle Cell Anemia

Rees, D. C. et al. Lancet Dec. 2010, 376, 201831.

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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
Frequent infection
Organ damage
High blood pressure
Extreme pain
Heart failure
Blocked blood vessels
2

Rees, D. C. et al. Lancet Dec. 2010, 376, 201831.

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Introduction

SCD Treatment
hiPSCs3
Human induced pluripotent stem cells
Some have problems differentiating
Not very reliable

Hydroxyurea4
Increased HbF levels
Carcinogenic
Works well or not at all

Bone Marrow Transplant

Risky
85% cured

More tests for better drugs

3
4

Sun, N.; Zhao, H. Biotechnology and bioengineering May 2014, 111, 104853.
Akinsheye, I. et al. en Blood July 2011, 118, 1927.
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Methods

Methods

DNA Extraction
Buccal Swab Protocol

DNA Isolation and HVI PCR Amplification

Gel Electrophoresis
If bands, alter master mix to get stronger bands
If no bands, either re-run PCR or alter master mix

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Results

Different Master Mix Concentrations

Table 1: Original Master Mix

Table 2: Altered Master Mix

Item

Item

Ingredient
dNTP
10x Buffer
Left Primer
Right Primer
Taq Polymerase
DNA
dH2 O

Concentration (l)
2.5
2.5
0.5
0.5
0.5
2.0
16.5

Ingredient
dNTP
10x Buffer
Left Primer
Right Primer
Taq Polymerase
DNA
dH2 O

Concentration (l)
2.5
2.5
1.0
1.0
0.5
2.0
15.5

*Note: DNA was added on a per tube basis

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Results

First Set of Primers

Figure 4: Left Primer

Figure 5: Right Primer

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Results

New Primers

Figure 6: Second Set of Primers

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Results

Gel Electrophoresis 1 & 2

Figure 7: DNA with Original

Concentrations of Master Mix, 25 l

Figure 8: DNA with Same

Concentration, 50 l

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Results

Gel Electrophoresis 3 & 4

Figure 9: DNA with Different Primer

Concentrations (0.5 and 1.0 l) with
Original DNA Sample

Figure 10: Stock Solution with 0.5 and

1.0 l Concentration of Reordered
Primers
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Discussion

Discussion

DNA with primer only worked with the first 2 tries (7,8)

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Discussion

Discussion

DNA with primer only worked with the first 2 tries (7,8)
Changing concentrations of primer did not work (9)

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Discussion

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)

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Discussion

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

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Discussion

Discussion
Running the PCR with reordered primers did not work either (10)

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Discussion

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)

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Discussion

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
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