Biochemistry Lab Report: D1S80, Creatinine and ALP Activity

This biochemistry lab report details an investigation into several biochemical assays and analyses. The report begins with an analysis of D1S80 variable number tandem repeats (VNTRs) using PCR, including experimental setup, safety measures, and the aim to determine the number of repeats from cheek cell DNA, followed by gel electrophoresis and analysis. The report then explores the diagnosis of Fragile X syndrome, including its causes, symptoms, and the role of PCR in its diagnosis. Additionally, the report includes a creatinine assay to assess kidney function, detailing standards, calculations, and interpretations. The report concludes with an analysis of alkaline phosphatase (ALP) enzyme activity, including experimental procedures, calculations of enzyme activity, and reflection questions regarding the clinical significance of enzyme levels, along with relevant references.

Running head: BIOCHEMISTRY 1
Biochemistry
Name
Date of Submission

BIOCHEMISTRY 2
Instructional Activity
Name
Date of Submission

BIOCHEMISTRY 3
1. D1S80 copies?
The size of a human genome is about 6billion bps while the average size of nucleotide bp
is 660 g/mol.
DNA from a single cell is 6billion X 660g
=3.96 pg
Thus there are two target copies of the gene in every 3.96pg of DNA. In theory, the in
human genome, about 25 to 100 ng of DNA is used for PCR. In 25ng, the copies of
DNA are 25000pg/3.960 multiply by 2 = 12625 copies (in 25ml reaction volume)
2. Safety
Wearing gloves
The pipette tips were changed after every sample addition to a PCR tube
No student was allowed to set up anyone else’s PCR
Each student set up a PCR reaction that contained their individual isolated DNA
Small reaction volumes were used
3. Method
For the 25 microliter reaction:
Mgcl2 =1.0 microliters
dNTPs= 4.0 microliters
buffer (Tris)=5 microliters
KCL= 0.25 microliters
Gelatin= 5.0 microliters

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4. Denaturation- At 95 degrees Celsius, the DNA template strands are heated to be
denatured so that the double strands separate into individual single strands (Tateishi-
Karimata & Sugimoto, 2014).
Annealing- at 65 degrees Celsius, the primers are allowed to bind to the template single
strand DNA strands to initiate replication.
Extension- at 72 degrees Celsius, the PCR protocol is allowed to repeat in a number of
cycles usually 20-30cycles to make so many copies of the targeted DNA region.
5. Aim: I hoped to determine the number of variable number repeat sequences isolated from
the DNA of cheek cells.
6. General strategy
To achieve this, the cheek cells were collected, DNA isolated and the D1S80 region was
amplified and analyzed for the finger print patterns to be studied per student.
7. Gel image
8. Standard curve
0 20 40 60 80 100 120 140 160 180 200
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Absorbance
Time Of Reaction (s)
Absorbance @420nm
Trend line equation:

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R2=
Tables
Band Migration (mm) Size (bp)
Positive control
Allele 1
Allele 2
Determine the number of repeats
Estimate number of repeats
Homozygous or heterozygous? Heterozygous
Parents
Siblings DNA
Crime scene: This analysis would be enough to be used in a crime scene and paternity
testing because, the amplified region from the DNA was the variable region and hence
each class member obtained different results.
Fragile X syndrome:
Symptoms
1. Hyperactive behaviors, delayed talking in children, intellectual disabilities,
hypersensitivity to bright light and loud noise, seizures and presentation with autistic
behaviors like lack of eye contact maintenance (Kidd et al., 2014).
2. Cause: fragile X syndrome occurs due to mutations in the FMR1 gene. This is a duplication
mutation in which the CGG repeat segment of DNA is expanded in the FMR1 gene.
3. when the CGG is repeated so many times in this gene, the gene is silenced thus, there is no
production of FMRP protein which under normal situations is involved in the production
of other proteins for the synapse development.

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4. Since the proteins coded for by the FMR1 gene are involved in special synapse connection in
the nerve cells, this mutation leads to the deficiencies of such useful proteins for nerve
transmission.
5. this mutation causes the disruption of the roles of the nervous system causing the associated
symptoms of fragile X syndrome.
6. in PCR, once cells have been collected, the DNA is isolated and PCR performed using primers
specific for the FMR1 gene. The amplification of this gene makes it to be closely
analyzed for the presence or absence of CGG repeating units and hence the overall
diagnosis.
Creatinine essay
Standards unknown
1 ml
creatinine
stock
0 15 30 45 60 24 48 120
Creatinine 2.4 4.8 12
Urine 0 0 0 0 0 12 24 60
1. 30nmol
2. 12 to 120nmol
3. 30/5= 6
=0.6ml
4. 120ml
5. trend line equation: y=0.0103x-0.0008
R squared value: 0.9974
6. 12 nmol
7. creatinine levels:
Replicate 1: 12-120nmol

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Replicate 2: 0-45nmol
Further questions
1. from the creatine and creatine phosphate breakdown in muscles (Lin et al., 2013).
2. they are filtered from the bloodstream by healthy kidneys.
3. urinalysis and glomerular filtration rates.
4. bilirubin- is a component in bile. Its presence in blood could be indicative of jaundice.
Ketones- due to fats metabolism- indicative of ketonuria.
5. to compare the readings of unknown sample from a standard which has known reference
ranges
6. drugs that are nephrotoxic
7. positive control- a sample which has been previously confirmed to have high creatinine levels.
Negative control- sterile distilled water.
8. impaired kidney functions.
Part C
0 20 40 60 80 100 120 140 160 180 200
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
f(x) = 0.00326117216117216 x + 0.131340659340659
R² = 0.999732671932119
Absorbance
Time Of Reaction (s)
Absorbance @420nm
activity:

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=300 U/ sec
=0.000165 U
Linear activity time: 0 to 180 seconds
Slope= 0.0033/min
Calculated activity: 295 U/sec
Serum
0 20 40 60 80 100 120 140 160 180 200
0
0.2
0.4
0.6
0.8
1
1.2
f(x) = 0.00457875457875458 x + 0.19021978021978
R² = 0.999955406788643
Absorbance
Time Of Reaction (s)
Absorbance @420nm
Activity= 180/0.8 = 225 U/sec
Volumes used:
Linear:
1st serum- 40-90 seconds
2nd serum- 0-60seconds
Slope:
1st serum: 0.0046/min
2nd serum: 0.9998/min
Rate of reaction:

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1st serum- 225U/sec
2nd serum- 1285 U/sec
ALP activity in undiluted sample:
1st serum- 12microliters
2nd serum- 5 microliters
Reflection questions
1. The enzymes can make it possible to measure the extent of liver function or damage.
2. – alanine transaminase
-aspartate aminotransferase
3. enzyme activities: creatine phosphokinase
Timeframe: hours after heart attack onset
4. for the purposes of quality control, that is, to make a comparison of the results.
5. so that all the enzymes can be bound to the enzymes at equilibrium.
6. high ALP means that the liver and gall bladder have functional problems (Malo, 2015).
Low ALP is rare but could be due to malnutrition.

BIOCHEMISTRY 10
References
Kidd, S. A., Lachiewicz, A., Barbouth, D., Blitz, R. K., Delahunty, C., McBrien, D., ... & Berry-
Kravis, E. (2014). Fragile X syndrome: a review of associated medical problems.
Pediatrics, 134(5), 995-1005.
Lin, Y. C., Bansal, N., Vittinghoff, E., Go, A. S., & Hsu, C. Y. (2013). Determinants of the
creatinine clearance to glomerular filtration rate ratio in patients with chronic kidney
disease: a cross-sectional study. BMC nephrology, 14(1), 268.
Malo, M. S. (2015). A high level of intestinal alkaline phosphatase is protective against type 2
diabetes mellitus irrespective of obesity. EBioMedicine, 2(12), 2016-2023.
Tateishi-Karimata, H., & Sugimoto, N. (2014). Structure, stability and behaviour of nucleic acids
in ionic liquids. Nucleic acids research, 42(14), 8831-8844.