SCD Presentation

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


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


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


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


5/23

Introduction

Common HBBMutations


-thalassemia

Methemoglobinemia



6/23

Introduction

Common HBBMutations


-thalassemia

MethemoglobinemiaHemoglobin C


I d i


7/23

Introduction

Common HBBMutations


-thalassemia


Hemoglobin E


I t d ti


8/23

Introduction

Common HBBMutations


-thalassemia


Hemoglobin E

Sickle Cell Anemia


Introduction


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


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


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


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


13/23

First Set of Primers

Figure 4: Left Primer

Figure 5: Right Primer9 / 1 4


14/23

Results


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


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


17/23

Discussion

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

13/14

Discussion


18/23

Discussion


Changing concentrations of primer did not work (9)

13/14


19/23

Discussion


20/23

Discussion


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


21/23

Discussion

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

14/14

Discussion


22/23

Discussion


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


23/23

Discussion


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

Documents

SCD Presentation