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University of Santo Tomas

Faculty of Medicine and Surgery


Class of 2017

Biochemistry
John Mark Villena
Section D

Laboratory Reviewer 4
**Imagine that Experiments 11, 12 & 13 are parts of 1 big experiment on Polymerase Chain Reaction
Experiment 11 performed to isolate the samples we need - genomic DNA from cheek cells & hair follicles
Experiment 12 PCR Proper, where a portion of the isolated DNA sample the PV92 gene, is
copied/amplified/polymerized over and over again
Experiment 13 Agarose Gel Electrophoresis, where the PCR products are checked if they are indeed the expected
products we wanted to isolate OR if the Alu insertion on PV92 is present/not (explained below)
Experiment 14 wala to iba na, Vitamin C Saturation test J

Experiment 11: Isolation of DNA from Cheek Cells & Hair Follicles
Genomic DNA
o Can be obtained from any microorganism/plant/animal/human
o Contains a copy of every gene from the organism
o Some genes are present/absent in individuals
o Regions
Exons DNA segments which code for proteins
Introns non-coding segments, separate adjacent exons
PV92
o Target sequence of the experiment
o An intron located in Chromosome 16
o May/may not contain the Alu insert; some individuals have it, some do not. The purpose of the experiment
is to identify whether or not the Alu insert is present in PV92
Alu sequence
o 300 base pairs long specific to humans
o Short Interspersed Elements (SINEs)/transposable elements capable of moving to different locations within a
given genome
o Found in a non-coding region of your DNA, not diagnostic for any disease or disorder
o PV92 Alu Polymorphism some people have the Alu insert in their PV92, some dont. The experiment can
be used to directly measure human diversity at the molecular level, data obtained can be applied in
population genetics ie how many of us have it/dont?
Isolation
o Samples
Cheek cells: Epithelial cells lining the oral cavity
Obtained by rinsing the mouth vigorously
with 10mL 0.9% Saline solution for
30seconds
1mL will be used for the experiment
Hair follicle cells: Epithelial cells around the base of
the hair
Obtained from 2 hairs with noticeable
sheaths/large roots
Trimmed to at least 2cm long
o InstaGene Matrix
Made up of Cation Exchange Resin (negatively
charged, microscopic beads) that chelate or trap
cations out of solution
Mg2+ - cation required by DNAses (contained in lysosomes) as cofactor for their activity to digest
DNA
Since all Mg2+ cations are already chelated out of solution, DNAses will be inactivated ensuring that
the DNA we are trying to extract will not be degraded
o Pre-incubation
Cells suspended in the InstaGene matrix are incubated at 56 C for 10 minutes
Loosens the plasma membranes/connective tissue and release clumps of cells from each other
Increased temperature also acts to inactivate DNases, which will degrade the DNA template
o Incubation
After the pre-incubation, the cells are then placed into a boiling (100C) water bath for 6 minutes
Boiling lyses/ruptures cell membranes to release cellular contents including lysosomes which contain
DNAses (already inactive due to chelation & temperature increase) & the genomic DNA we need

University of Santo Tomas


Faculty of Medicine and Surgery
Class of 2017

Biochemistry
John Mark Villena
Section D

o Isolated DNA
After incubation, resuspend the contents. Pellet the matrix at 6,000xg for 5min to separate the
cellular components
Isolated DNA will be solubilized in the supernatant, the precipitate will be other insoluble
components of the lysed cells
Transfer the supernatant in another tube & store it in the InstaGene Matrix to make sure residual
DNAses are inactivated
Spectrophotometric quantitation of DNA
o 10ul of supernatant + 1mL distilled water
o Measure the Absorbance at 260nm & 280nm it is common for DNA to be contaminated with proteins, we
need to measure at both wavelengths to quantify how much DNA & Protein we have
260nm - peak absorbance wavelength of DNA
280nm peak absorbance wavelength of Proteins
o Obtain the A260/A280 ratio
%DNA %Protein
260/280
A measure of DNA purity
100
0
1.80
[DNA]/[Proteins]
95
5
1.79
1.80 pure DNA
90
10
1.78
Low ratio would indicate high protein contamination
70
30
1.74
o Calculate DNA concentration
1 A260 unit = 50ug of dsDNA/mL
If your A260 = 1, 1mL of your solution would contain 50ug of double stranded DNA
Example, you obtained an A260 of 0.320 ; x = unknown DNA concentration
1 : 50ug/mL = 0.320 : (x) ug/mL
x = (50 x 0.320) / 1
x = 16ug/mL dsDNA

Experiment 12: Polymerase Chain Reaction


In vitro system for DNA replication that allows a target DNA
sequence to be selectively amplified several million fold in
just a few hours
Uses
o Detection of hereditary diseases
o Identification of genetic fingerprints
o Diagnosis of infectious diseases
o Cloning of genes
o Paternity testing
Requirements
o DNA Template the isolated DNA from Experiment 11; provides the template for DNA replication
o PCR Master Mix
Taq DNA Polymerase
DNA-directed DNA Polymerase from Thermus Aquaticus (thermophile)
o Polymerizes in the 5'3' direction
o No 35 exonuclease activity for proof-reading
Optimum temperature: 7580 C making it heat-resistant & a viable enzyme to be used in
PCR as the experiment will reach expose the solution to very high temperatures
PCR Buffer salt buffer; maintains the optimum pH for the Taq polymerase & provides an ionic
environment for the reaction as Taq pol requires Mg2+ as a cofactor
Deoxynucleotidetriphosphates (dNTPs)
dATP, dTTP, dGTP, dCTP
Nucleic acid monomers which the polymerase use to add to the new/growing DNA daughter
chain
Primers
20-30 nucleotide segments of sequence-specific DNA fragments which bind to the flanking
sequences of the target gene (upstream) on each side
Pair of non-identical primers: Reverse & Forward
Signal the DNA polymerase where to start making copies
They're on each side on the segment so that the newly synthesized complementary pairs
can bind to each other.

University of Santo Tomas


Faculty of Medicine and Surgery
Class of 2017

Biochemistry
John Mark Villena
Section D

Steps
o Denaturation
Exposure of the double stranded DNA template to 94C for 1min
Causes melting by disrupting the hydrogen bonds between complementary bases yielding 2 single
stranded DNA strands

o Annealing
Reaction temperature is lowered to 60C for 1min allowing the primers to anneal/bind to the
flanking sequences on each single strand upstream the target gene
Annealing is affected by
Annealing temperature
o 3-5C below the Tm of the primers used
o Must be optimized first but for us, 60C is the annealing temperature
Complementarity between the primers & the DNA template
o Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very
closely matches the template sequence
Taq Polymerase shown in blue circles, recognizes the double-stranded substrate and binds to the
3 end of the primer

o Polymerization
Reaction temperature is raised to 72C for 2min as this is the optimum temperature of Taq
Polymerase for it to begin extension/DNA replication
REMEMBER: Polymerization only occurs in the 53 direction!

GeneCycler/MyCycler thermal cycler


o PCR Machine, settings used: 3-steps in Cycle 2 for 40x (3.5hours)
o Final cycle ensures that the final extension reaction goes to completion & all PCR products are made
o Amplification
Amplification in PCR is exponential
Amplification product (amplicons) = 2^n cycles = 2^40 = 1,099,511,627,776 copies of the target
gene

University of Santo Tomas


Faculty of Medicine and Surgery
Class of 2017

Biochemistry
John Mark Villena
Section D

Experiment 13: Agarose Gel Electrophoresis


Standard method used to separate & identify nucleic acids
o Molecules will be separated based on their molecular size & net charge as they
migrate in a gel under the influence of an electric field
o When charged molecules are placed in an electric field, they migrate toward either
the positive or negative pole according to their charge
Anode (+) charged pole where (-) charged particles migrate to
Cathode (-) charged pole where (+) charged particles migrate to
DNA Migration towards the anode (+) is influenced by
o Negatively charged phosphate backbone
o Size of the DNA fragment
The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can
move more easily than larger ones
Larger fragments will migrate slower & lag behind while smaller fragments will migrate faster toward
the anode
Fragments of the same size stay together and migrate in what appear as single "bands" of DNA in
the gel

2 types of Gel Electrophoreses

Origin
Composition
Gaps within the gel
Resolving power
Range of separation
Preparation
Direction
Type of molecule it
separates

Polyacrilamide Gel
Electrophoresis
Polymer of acrilamide
Lots of molecules held
together by intermolecular
forces
Smaller
High can separate

Agarose Gel
Electrophoresis
Polysaccharide from
seaweed
One large molecule
Larger
Low can separate

fragments that differ by 1


nucleotide
Small up to 1,000 base
pairs long
Difficult, acrylamide is a
potent neurotoxin

fragments that differ by


20+ nucleotides
Large up to 200
50,00bp

Vertical
Protein > DNA

Horizontal
DNA

Easy

Experiment proper
o Amplified DNA products will be separated via electrophoresis to make sure that we indeed isolated the
fragment of interest by comparing the samples to standards of known molecular weight
o 8 wells where APPROPRIATE standards &
samples should be placed
o Set to 100V & electrophorese for 30min
o Tracking dye
Bromophenol blue in 50% Glycerol
solution
Stains the colorless DNA bright blue
Dye used to track the progress of the
DNA through the gel
o Gel Documentation Aparatus
Used for the imaging and documentation of nucleic acid suspended within agarose gels
Gels are typically stained with ethidium bromide or other fluorophores such as SYBR Green
A gel doc includes an ultraviolet (UV) light transilluminator, a hood to shield external light sources
and protect the user from UV exposure, and a camera for image capturing

University of Santo Tomas


Faculty of Medicine and Surgery
Class of 2017

Biochemistry
John Mark Villena
Section D

o Ethidium Bromide
Previous gold standard as a DNA visualizing agent
Fluoresces to red-orange or pink under UV light
Moves into the hydrophobic environment between the base pairs of DNA
Removal of water molecules: ethidium bromide will fluoresce
Unsafe to use
Toxic and a potent carcinogen, it intercalates dsDNA by inserting itself between the strands
Increased absorption in the skin and mucus membranes
Causes irritation to the eyes, skin and respiratory tract
SOP
Protective Clothing: fully-buttoned lab coat, long pants and closed-toe shoes.
Eye Protection: safety glasses with side shields at all times
Gloves: disposable nitrile gloves
Fume Hood
o Fast Blast DNA
Thiazine dye containing cationic compounds which tag negatively charged phosphate groups on
DNA staining it deep blue on Agarose Gels
Provides inexpensive & safer method of documenting the results of DNA electrophoresis
Take a longer time than fluorescent DNA stains such as ethidium bromide that give rapid result
Can also be used as a tracking dye
o UV Trans illuminator
Used in visualization of nucleic acids stained with uorescent dyes such as ethidium bromide and
other dyes (acridine orange)
Emits a short wave UV light can cause severe damage even with very short exposure
SOP
Never operate without the safety hood or cover in place when capturing gel images
Always wear protective equipment such as gloves, face shields, and long sleeves lab coats
Use thick gloves such as thick nitrile or latex gloves
Keep away from escaping rays
Damage it may cause
Photokeratitis - Inflammation of the cornea
Eye injury - can occur due to very brief exposure or a flash of intense UV
Erythema - can occur within a few seconds of exposure to a concentrated form of UV
Prolonged exposure causes premature aging and cancer of the skin
Expected result
o PV92
Target gene that has been amplified
Dimorphic due to the presence/absence of the Alu insert so
there are two possible PCR products
641 bp (PV92 w/o Alu)
941 bp (PV92 w/ Alu)
Alu insert 300 base pairs long
o Interpretation of 3 possible Genotypes
Homozygous Positive (+/+)
AGE result should only show 1 distinct band at the level of 941bp
Look at Lane 2, you can only see 1 fat band near 941bp meaning that person only has the
Alu insert in both copies of his Chromosome 16
Homozygous Negative (-/-)
AGE result should only show 1 distinct band at the level of 641bp
Look at Lane 3, you can only see 1 fat band near 641bp meaning that person does not
have Alu insert in both copies of his Chromosome 16
Heterozygous (+/-)
AGE result should show 2 distinct bands at the level of 941bp & 641bp
Look at Lane 4, you can see 2 bands, the band near 941bp represents the allele with the
Alu insert while the band near 641bp represents the allele without the Alu insert making
that person Heterozygous

University of Santo Tomas


Faculty of Medicine and Surgery
Class of 2017

Biochemistry
John Mark Villena
Section D

Experiment 14: Vitamin C Saturation Test


Vitamin C
o L-enantiomer of ascorbic acid - 2-(1,2-dihydroxyethyl)-4,5dihydroxy-furan-3-one
o Water soluble vitamin
Owing to its many OH groups which imparts hydrophilic
properties
Absorbed in actively in the jejunum of the SI
Transported freely in the plasma/bound to albumin
Excreted via the urine
o Anti-oxidant
Easily oxidized to DHAA by heat, light, and oxygen. DHAA is easily reduced again to AA
Further oxidized to Gulonic Acid
Potent vitamin against free radicals & ROS
Readily donates electrons to break the chain reaction of lipid peroxidation (antioxidant)
Tocopherol and glutathione also rely on AA for regeneration back to their active isoforms
o Hexose derivative
Synthesized from glucose & galactose
Synthesized by plants & some animals
Via a modified Uronic Acid Pathway - a minor
glucose metabolic pathway
Humans lack the enzyme L-Gulonolactone oxidase
which proceeds to Vitamin C synthesis
Failure to synthesize this vitamin makes it essential hence
we must obtain it from the diet
o Uses
Enhances absorption of iron
Blocks degradation of ferritin to hemosiderin
Coenzyme and cofactor in the following processes:
hydroxyalation of proline in collagen synthesis
oxidation of PHE-TYR
conversion of folacin-THFA
conversion of tryptophan to 5-OH tryptophan & formation of serotonin, E and NE
hydroxylation of certain steroids in the adrenals
Resistance to infection
Production of interferons
Maintenance of integrity of membranes
Synthesis of bile acids
o Recommended Nutrient Intake (RNI):
Filipino male: 75 mg
Filipino female: 70 mg
Pregnant mothers: 80 mg (18 years old and below); 85 mg (19 years old and above)
Lactating mothers: 115 mg (18 years old and below); 120 mg (19 years old and above)
o Deficiency
Scurvy; mainly affects older, malnourished adults
Signs and Symptoms due to malformation of collagen
Dry and splitting hair
Gingivitis
Bleeding gums
o Excess
GIT: osmotic diarrhea
Transformed in the body to oxalate (kidney stones)
Excess ascorbate in the urine and feces can falsify lab tests such as glucose in the urine and fecal
occult blood test
Accelerated thickening of the walls of the big arteries in the neck

University of Santo Tomas


Faculty of Medicine and Surgery
Class of 2017

Biochemistry
John Mark Villena
Section D

Experiment
o Quantification of the Ascorbic Acid excreted in the urine in response to a test dose of 500-1000mg
In individuals with low Vitamin C reserves, it is held in the tissues & the total excretion is less than
26% of the test dose in 24 hours
Increased intake of Vitamin C Increased excretion
o 2 groups
Experimental
6 consecutive days starting one week prior to experiment - 100 mg PO daily
One day prior to experiment - 500 mg
Control
One day prior to experiment - 500 mg
o Urine collection
One day before experiment 24-hour urine collection test with toluene (preservative)
Measure total volume then collect 250 mL of urine for vitamin C content determination
o Modified Titrimetric Method of Bessey
Used to quantify urinary vitamin C
Depends on the reduction of the dye (2,6-dichlorophenol-indophenol) to a colorless compound by
vitamin C
o 10% Metaphosphoric Acid
Used to acidify the urine
Maintain the proper acidity for the titration reaction
Maintain a low pH to prevent auto-oxidation of ascorbic acid at high pH
o Titration method
Acidified urine in the biuret added dropwise to
1mL 0.02% 2,6 dichlorophenol-indophenol - redox indicator dye
Basic pH: blue initial color without urine addition
Acidic pH: red/pink since we acidified the urine we would expect this color upon urine
addition
Vit C present in the urine will reduce the reagent to its colorless form
In the process, Vit C is oxidized to DHAA

Titration Equivalence Point theoretical point where 1mL of the dye has completely been reduced
by an equivalent amount of Vitamin C
Experimental
o Would require less volume of urine to reach the equivalence point because their
urine has higher vitamin C concentration
o High urinary vitamin C output because their body reserves are high due to prior
intake (100mg per day/6 days) of vitamin C
Control Group
o Would require more volume of urine to reach the equivalence point because their
urine has low vitamin C concentration
o Low urinary vitamin C output because the natural reserves of Vitamin C are not
high enough to reach a spill-out point
Titration End Point observable change brought about by the equivalence point, we perceive this
as the disappearance of the dyes red color
o Computation