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Running head: GENOMICS
Name of the Student:
Name of the University:
Running head: GENOMICS
Name of the Student:
Name of the University:
The basic functional and physical unit of heredity are genes, which are composed of
DNA (deoxyribonucleic acid) 1. Segments of DNA are known as genes. Any change in the
sequence of nucleotides or changes in gene expression can cause cancer 4.
Genes such as TAL1 and CDH1 are linked to cancer 5. Normal as well as malignant
hematopoiesis involves t he transcription factor TAL1 which stands for T -cell acute
lymphoblastic leukemia ( [T-ALL ] 1). TAL1 requirement is essential during the developmental
process, during the survival of adult hematopoietic stem cell, its quiescence stage and during
its maturat ion 2. A classical cadherin is also encoded by CDH1 (Calcium - Dependent Adhesion
Protein). Alternate splicing of this gene produces variants of the transcript 6.
When the gene promoters are methylated, the gene copy gets affected as the methylated
groups act as a barrier that protect s the promoter and prevent s the DNA polymerase to attach
to the promoter 3. An example of epigenetic domination is methylation. In the methylation assay
conducted on DNA obtained from CDH1 and TALL1 , Hpa ll was used which is a restriction
enzyme that is sensitive to methylation. The level of methylation that is present in the digestion
is analysed by Hpa ll. Since Hpa ll does not cut at sites that are methylated, hence during a qPCR
run the increased rate of m ethylation is directly linked to an increased volume of product
To observe how TALL and LEPI cell samples affect the rate of DNA methylation and
how DNA methylation affects the rate of gene expression an experiment was performed. It was
hypothes ized that huge differences would be observed .
In this experiment conducted, three samples of gDNA – Hpa ll digested , Msp l digested,
and undigested - were taken, the total weight of which was 100ng with a volume of 15 µl. 2
samples of each type of the gDNA were taken to which future primer MSRE was added. All
the samples, total 6, were amplified which produced 12 PCR reactions. They were prepared in
0.2ml of 8 well strips. To each well, 2 µl of sample containing diluted gDNA was added. The
tw o reaction mixtures that were prepared contained SYBR and a pair of primers.
This experiment comprised of two parts: total conversion of RNA into cDNA, and
qPCR analysis of gene expression.
The experiment utilised the final concentration of ng/ul for Lepi and TALL cells and
also a SensiFAST cDNA synthesis kit. To synth esi ze cDNA, a reaction volume of 20 µl
comprising of 1x concentration of TransAmp buffer and 1 µl of reverse transcriptase enzyme
was needed. The components of qPCR comprised of 1x concentration of SensiFast SYBR and
0.2 µM of forward and reverse primers , the total volume of which was 8 µl . NFYB analysis as
a normal calculation was also performed.
In this experiment, sequencing and analysis of a pa rticular amplicon was performed.
The first step was to isolate and amplify the sample by performing a PCR. The PCR
components included: genomic DNA of 1.5ng/ µL, Taq mix of concentration 1x, forward and
reverse primer mix of 0.5 µM . The final volume of the reaction mixture was 50 µl . After PCR,
the amplicon was ligated to each end of the oligonucleotide adaptors that were double stranded
via DNA end repair and ligation. The components of DNA end repair comprised of DNA
fragments that were 0.5 - 5 µg. The reaction mix of 5 µl and enz yme mix of 2.5 µl makes a final
volume of 50 µl . This mixture was then incubated at 20 °C in a heating block for 10 minutes.
After this, the reaction mixture comprising of PCR amplicon of 100ng, sequencing adaptor of
0.75 pmo les, rapid ligation buffer of 1x concentration and DNA ligase 1 unit was prepared.
The total volume of the mix was 20 µl and this reaction mix was utilised for ligation of
sequencing adaptor of the PCR amp licon. Then the adaptor ligated sequences were fed to the
PCR and extension was performed. The PCR mix comprised of 1:100 PCR amplicon, Taq
polymerase of 1x concentration and PCR primer of 0.5 µM. The total volume of the reaction
mix was 50 µl .
MSRE LEPI 1 and 2
Figure 1: the CT value differences are shown for Lepi MSRE using restriction enzymes Hpall
and Mspl minus the no enzyme control in Lepi 1 and 2.
12.00 HpaII vs MspI CT values in LEPI Samples
CT (MSPI) - CT (NO
CT (HpaII) - CT (NO
MSRE_3_LEP1 MSRE_4_LEPI1 MSRE3_LEPI2 MSRE4_LEPI2
MSRE TALL 1 and 2
Figure 2: CT values differences for TALL MSRE using restriction enzymes Hpall and Mspl
minus the no enzyme control in samples of Tall 1 and 2.
LEPI qPCR Gene Expression
Figure 3: relative fold difference is shown in gene expression for two samples of LEPI 1 and 2
Hpall vs Mspl CT values in TALL samples TALL_1_MSRE_4
CDH1_1 CDH1_2 avergare relative fold difference in gene
Gene Expression in LEPI sample 1 vs 2
TALL qPCR Gene Expression
Figure 4: relative fold difference is shown in gene expression for two samples of TALL 1 and
Figure 5: Nanopore agarose gel. In the gel shown above, first column is the hyper ladder and
ha s a number of b ase pair s beside it. From practical 1, Lane 1 shows CDH1 PCR amplicon and
lane 4 shows TALL1 PCR amplicon. From practical 2, lane 2 shows CDH1 sequence ada ptor
ligated PCR amplicon, lane 3 shows CDH1 sequence adaptor No ligase PCR amplicon, lane 5
shows TALL1 sequence adaptor ligated PCR amplicon and lane 6 shows TALL1 sequence
adaptor no ligase PCR amplicon.
avergare relative fold difference in gene
Gene Expression in TALL Sample 1 vs Sample 2
The objective of the experiment was to observe any fold difference in the two cancer
cells LEPI (CDH1) and TALL (TAL1). In the results obtained, figure 3 showed CDH1 (LEPI)
with an exceptionally low average of 0.25627 and a standard deviation of 9.295339 whereas
sample 2 showed an avera ge of 27.7051 which was comparatively much higher than that of
sample 1, and a standard deviation of 22.96878. The difference between average and standard
deviation calculated for LEPI sample 1 was low but that for LEPI sample 2 was significant ly
high . Res ults obtained for TALL (TAL1) samples were also similar. A low average of 0.98 for
sample 1 was observed with a standard deviation of 0.210516 whereas for sample 2 an average
of 2.668985 and a standard deviation of 0.53376 was observed. The difference calc ulated
between average and standard deviation of TAL1 sample1 was low but that of TAL1 sample 2
was significantly high.
In the MSRE assay experiment where genomic DNA was digested with Hpa ll or Mspl
for qPCR, the figure 1 indicated samples of MSRE 3_Lepi1& 2 and MSRE 4_Lepi1&2. The first
column indicated the average of MSRE_Lepi_1 for CT (MSP -1)-CT (No enzyme) as 6.73 with
a standard deviation of 1.23137024. The second column indicated an average of
MSRE 4_Lepi_1 as 6.87 and a standard deviation of 1.25650024. The third column indicated
an average of MSRE3_Lepi_2 as 8.13 and a standard deviation of 2.46939895. The last and
the fourth column indicated an average of MSRE4_Lepi_2 as 8.30 and a standard deviation of
2.5197948. Columns 5,6,7 and 8 showed CT values of Hpall without the CT value and no
enzyme. In the 5 th column, an average of MSRE3_Lepi_1 as -0.65 was ob served.
Samples of TALL_1_MSRE_4 &5 and TALL_ 2_MSRE_4 &5 were observed in figure
2. The first column indicated an average of TALL_1_MSRE_4 as 5.29 with a standard
deviation of 4.421440633. The second column indicated an average of TALL_1_MSRE_ 5 as
5.36 with a standard deviation of 4.479676426. The third column indicated an average of
TALL_2_MSRE_4 as 6.86 with a stan dard deviation of 1.644769881 and the last column that
is the fourth column indicates an average of TALL_2_MSRE_5 as 6.72 with a standard
deviation of 1.612519491.
A type of polymerase chain reaction is real time quantitative PCR during which
monitoring o f the synthesis of desired DNA product occurs during real time. A fluorescent dye,
SYBR Green is used during the qPCR reaction . It emits a signal that upon combining with the
DNA which is newly synthesized turns fluorescent . The initial amount of DNA that was used
as the starting material was quantified by plotting the degree of fluorescence detected with each
PCR cycle. To quantify the level of gene expression of TAL1 and CDH1 genes, qPCR was
Inability to meet the goals of the experiments and inaccurate results obtained for all
qPCR experiments can be attributed to ussies with the protocol or with the samples used. To
identify, abolish or reduce errors, both technical as well as biological repl icates could have
been run so that the reliability of the protocols as well as that of the cells could be tested,
respectively. Technical replicates utilises the same sample for multiple rounds to test the
investigation protocol. Biological replicates util ise different samples to test viability of the
sample. The statistical analysis of the results obtained co mprised of calculating averages and
standard deviations of qPCR gene expression and methy lation results. Large standard
deviations were found with all results. Abhorrent data was not considered in the analysis and
was eliminated which resulted in low standard deviation values . However since some standard
deviation values obtained were larger than the averages obtained, hence this indicated
inaccurate an d unreliable results. Thus as mentioned above , performing both types of
replicates - technical and biological , could have reduced the deviation of results obtained and
improved the outcomes.
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