HBB GENE ANALYSIS

EMILY CRIBAS

Sickle cell anemia is directly caused by a mutation in the HBB gene. The exact sequence of the
gene is shown1 . The purpose of this project was to successfully collect, amplify, and sequence this
gene for two different human subjects. This was done through a buccal swab protocol, several
polymerase chain reactions (PCR), and eventually, gel electrophoresis to visually represent the
successful amplification of the gene, as well as its length.
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 1. GenBank HBB Sequence

The DNA samples were collected using the buccal swab protocol. The gene was amplified
using PCR, and the gel electrophoresis showed whether the gene actually amplified at the right
base pair length. Before running PCR, the GenBank sequence was used to determine which
region of the gene to amplify. In this case, we used both exons 1 and 2 with the intron region
between the two included, and we later used just exon 3. To narrow down the amplified region
to just this portion of the gene, we used primers. Two sets of primers were needed for the first
amplification region because it contained two exons. The primer output from a website, Primer3,
is shown below2,3 . The left primer for exon 1 was used, and the right primer for exon 2 was
used in this case. The primer output for exon 3 is also shown below4 . Overall, when bands on
the gel were not present, the master mix used to place in each PCR tube was altered and PCR
was rerun. If bands were present, the same thing was done to make bands stronger. Once the
bands were strong enough, the gene was sequenced to show if the subjects had the appropriate
mutations or not.
Overall, this project was run over the course of 12 weeks with no results substantial enough
for sequencing.
Date: December 7, 2014.
1

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Figure 2. Primer3 Output for Exons 1

Figure 3. Primer3 Output for Exon 2

Figure 4. Primer3 Output for Exon 3

For the first couple of weeks, DNA samples were collected using the buccal swab protocol.
The samples varied from 40-55 ng/ml in concentration using the NanoVue Spectrophotometer.
After these samples were collected and isolated, PCR samples were made using a master mix
that included DNA on a per tube basis. 23 l of this mix was added to each tube, with 2 l
of DNA added to each one, totaling 25 l. The master mix first used is highlighted in Table 1.
After this master mix was run through PCR, two tubes for each subjects DNA were placed in
wells for gel electrophoresis, along with a DNA ladder, and a control that contained no DNA.
Results from the gel are shown.5 The gel showed strong bands for only one subjects DNA, but

HBB GENE ANALYSIS

the length of the DNA read about 2 Kbp, while the gene portion should have read about 500
bp.
Table 1. Original Master Mix
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

Figure 5. Gel Electrophoresis

The week after, only one subjects DNA sample was used, this time using 50 l. Bands
were visible, but not as strongly, and not at the right base pair length. The gel showed DNA
samples 1 Kbp in length6 . After trying to re-extract DNA samples from the subject, no bands
appeared from, this time, both samples. For the next week, the master mix was edited to
increase concentrations of primers from 0.5 l and 1.0 l and reduce dH2 O concentrations by 1
l. The gel obtained from these PCR results also had negative results. The process for the next
PCR setup is the same, except this time, stock DNA was used to ensure whether the issue was
degraded DNA or something else. 6 PCR tubes were set up: 2 filled with 1 l stock DNA and
the original master mix, 2 with one subjects DNA samples and master mix, and 2 with subject
DNA and increased primer concentrations. The results of this PCR setup was shown in Figure
7.7
The last alteration that was made was increasing the primer concentration once again, this
time using only 2 l stock DNA to add after. Along with this, consistent negative results using

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Figure 6. Gel with One DNA Sample

Figure 7. Stock & Subject DNA

primers for exons 1 and 2 led to the idea that the primers might be the issue. The results of
this PCR setup is shown below8 . The ladder showed up very distinctly, while the stock DNA
appears streaked. This may be due to some degradation of the DNA. Since the stock was most
likely not the problem, however, the main reason PCR might not have worked could be because
the primers formed dimers or degraded (by multiple freeze-thaw cycles) or the exon region was
too small to amplify. Overall, there are many potential reasons as to why PCR did not work
and most of them point toward a problem with the primers, while some can point towards using
cheek swab samples versus blood samples.

Figure 8. Gel with Reordered Primers