A.

Introduction
Polymerase chain reaction (PCR) is a technique used to produce high yields of specific
DNA target sequences. PCR was developed in 1983 by American biochemist Kary Mullis.
Molecular biology uses PCR to make copies of small fragments of DNA or RNA. PCR makes it
possible to generate millions of copies of a particular sequence of DNA from a small amount.
PCR is used for sequencing, detecting the presence or absence of a gene, helps identify
pathogens, and is used in forensic science. PCR uses thermal cycling which is a process that
involves heating and cooling down the reaction. A current application of this technique is to
test for COVID-19. In this case the PCR test for COVID-19, looks for genetic material of SARS-
CoV-2. They use this technology to amplify small amounts of DNA, which is replicated until
SARS-CoV-2 is detectable if present. This technology has really impacted this area of COVID-19.

I. Purpose
The purpose of this experiment is to isolate DNA collected from epithelial cells. The DNA
will be amplify using PCR. Two modes of gel electrophoresis will be used for analyzes.

II. General Plan

Genomic DNA will be isolated from epithelial cells. The cells are going to be broken
open. With the use of guanidine salts the DNA is going to bind to the magnetic core beads.
Using the beads, we are going to purify the DNA. Using an elution buffer, the DNA is going to be
collected. PCR will be done to amplify the DNA. Analyzes will be performed using
polyacrylamide and agarose gel.

III. Theory
Isolation of DNA
A lysis buffer is used on the epithelial cells that were collected from the cheek. The lysis
buffer contains the following: EDTA, dithiothreitol (DTT), detergent, and guanidine. EDTA helps
reduce protease activity. DTT it’s a redox reagent that is used to stabilize DNA, it prevents it
from being oxidized or reduced, and helps break down the epithelial cells. The detergent is
used to also help break open the cells. Guanidine is used to form a salt bridge between DNA
and an iron core bead. When a magnet force is applied these iron core beads allow us to
further purify our DNA from unwanted proteins. A wash buffer is used to remove any proteins
that are still binding to the DNA. To elute the DNA from the beads an elution buffer is used. The
elution buffer does not contain guanidine. Since no guanidine is contained in the elution buffer
the DNA is able to fall off the beads.

Oligonucleotides synthesis
To chemically synthesize single stranded oligonucleotides or DNA, solid phase chemistry
is used. Solid phase chemistry uses a solid support to start the process. In this case that solid
support is a Controlled Pore Glass bead (CPG) figure 1. The CPG bead is going to have its base
attached to it through a linker group known as syccinylaminohexanoylaminopropyl. The 5’-OH
ends of each of the deoxyribonucleosides has a chemical blocking group known as dimethoxy
trityl (DMT). This blocking group prevents the 5’-OH ends from reacting with anything until
ready for the next base to be added.
 Figure 1: DMT protected nucleoside attached to the controlled-pore glass (CPG)

DNA is made out of four bases: adenine, cytosine, guanine, and thymine. These bases are going
to be added one at a time in order to grow our molecule. The first step begins by removing the
DMT which protects the 5’-OH with trichloroacetic acid (TCA). This process is known as
detritylation in which the 5’-OH is freed up, making it ready for reaction to start. After, the
nucleoside of choices is brought in order to produce the sequence of choice Tetrazole and
phosphoramidite are mixed together and added into the reaction. The phosphate that is
attached to the phosphoramidites will add on to the reactive oxygen 5’-OH from the step
before. This means that the second base has been added carrying its protective group on it. If
the improper base is added after the coupling step acetic anhydride is added. This is known as
capping, in which the reaction is stopped and nothing else can be added. If the base was added
properly acetic anhydride has no effect and the reaction continues. The trivalent phosphorus
linkage formed in the coupling step is converted to the stable, pentavalent phosphorus linkage
of biologically active DNA. This is an oxidation step that uses iodine. The cycle repeats until an
oligonucleotide of interest is made. After the oligonucleotide is assembled, a deprotecting step
is done with ammonium hydroxide. This step removes the phosphate protecting groups,
separates the oligonucleotides from the solid support, and removes the protecting groups on
the bases. Chromatography is used to separate by sizes unfinished oligonucleotide chains.

Polymerase Chain Reaction (PCR)

PCR contains the following: Buffer, MgCl2, dNTPs, Primers, template DNA, autoclaved
H2O, and Taq polymerase. Buffer is used to help maintain pH. Certain enzymes work best at a
particular pH. Enzymes need divalent cations in order to work. In this case the divalent cation is
MgCl2. The MgCl2 binds with the dNTP and makes a 3D shape that is recognized by the enzyme
when it’s going to add the dNTP onto the next base in the reaction. PCR primers are
oligonucleotides, typically 15-30 bases long. When choosing primers, it is important that they
not contain bases complementary to themselves or with each other. Templates could be either
single stranded or double-stranded DNA or RNA. The enzyme that is used for PCR is a Tag DNA
polymerase. This will start the reaction. Amplification of DNA is achieved by the following three
steps also known as thermal cycling:
 1. Denaturation: Denaturing allows the double-stranded DNA template to be
heated in order to separate the two strands of DNA. DNA denaturation is a critical step.
The practical range of effective denaturation temperatures for most samples is 94 C to
96 C. Anything higher could degrade DNA
2. Annealing: Annealing allows the primers which are short DNA molecules to bind
to flanking regions of the target DNA. The annealing temperature is based on the
melting temperature Tm of the oligonucleotides chosen for PCR amplification. The
annealing temperature is raised in 2 C to 5 C increments.
3. Extension: Extensions allow the DNA polymerase to extend the 3-prime end of
each primer along the template strands. Primer extension occurs effectively at a
temperature of 72 C and seldom needs optimization. This technique may enhance
overall yield. Typically, 25-45 cycles are required for extensive amplification of the
target.

The Hot Start technique is used so that the reactants do not mix until reaching a temperature
high enough to suppress primer annealing to non-target sequences. This is done because a
major obstacle to PCR amplification appears to be competing side reactions such as the
amplification of non-target sequences in the background of DNA (mis-priming). This usually
happens during the pre-PCR setup, when all the reactants have been mixed, usually at room
temperature right before thermal cycling is started

Polyacrylamide vs Agarose gel

Polyacrylamide and agarose gels are used in this experiment to compare the ability to
separate various sizes of DNA. Polyacrylamide gels are able to separate 10 to 800 bp. Things
that fall into this category include small RNAs, small DNA restriction fragments, and small
sequencing fragments. Agarose gels are able to separate 300 to 50,000 bp. Things that fall into
this category are large RNA’s and large DNA restriction fragments. Within both categories of
gels there could be either native gels or denaturing gels. Native gels might be double stranded
DNA (dsDNA) or RNA in a native configuration. This gel is done with normal salt buffers, for
example tris borate. Native gels separate based on both charge and size, while allowing
biomolecules to retain their structure. In a denaturing gel you can either have single stranded
DNA (ssDNA) or RNA that is separate based on size. B-mercaptoethanol for SDS is used in
denaturing gel to coat the protein with negative charges and disrupt the structure. This gives it
a constant charge-to-mass-ratio, thus depending only on size for separation. This gel could use
NaOH if its DNA or formaldehyde if its RNA.

Short Tandem Repeats (STR’s)

Short Tandem Repeats (STR) analysis is a method used to compare allele repeats loci in
DNA. STR’s can be use in PCR in order to amplify the DNA. The length of STR’s are around 2 to 8
base pairs that repeat multiple times in the DNA sequences. STR loci are targeted with
sequence-specific primers and amplified using PCR. The resulting fragments are separated and
detected using electrophoresis.
B. Flow Chart
Isolation of DNA using the DNA-IQ system
1. Preparation of lysis buffer. For each sample isolated, pipet 350 uL of lysis buffer into 1.5
mL snap cap tube. USING A NEW AUTOCLAVED PIPET TIP. Add 3.5 uL of 1 M DTT to each
350 uL sample buffer. Mix by inversion
2. Remove foam swab from sterile wrapping. Collect buccal cells
3. Place inside a 1.5 mL tube. Snap off plastic stem above the foam pad
4. Add 250 uL of prepared lysis buffer + incubate at 95 C for 30 mins. Remove the tube and
vortex briefly.
5. Transfer the lysis buffer to a new tube. Squeeze excess liquid using 1 mL pipet tube. Add
any recovered liquid to the lysis buffer. Briefly spin the tube containing the foam pd in a
microfuge, and immediately collect any liquid in the bottom of the tube. Added to the
same tube.
Side note: Failure to promptly collect the liquid in the bottom of tube will result in the liquid
being soaked again.
6. Vortex stock resin bottle for 10 seconds. New autoclaved tip (remove) 7 uL of the resin
add it to the 250 uL of prepared lysis buffer. Keep resin suspended until just before
pipetting it out
7. Vortex sample/lysis buffer/resin for 5 sec and place the tube in the magnetic stand.
Separation occurs instantly.
Side note: if resin doesn’t from a distinct pellet on the side of the wall, vortex place back on
stand.
8. Remove/discard all of the solution without disturbing the resin on the side. Gently expel
resin back into tube to allow reseparation.
9. Add 100 uL of prepared lysis buffer to the tube containing the resin. Remove the tube
from magnetic stand and vortex for 2 seconds at high speed. Return to magnetic stand
and discard all lysis buffer.
10. Add 10 uL of 1x wash buffer. Remove tube from the magnetic stand and vortex for 2 sec
11. Return to magnetic stand and discard all wash buffer. Repeat 12 and 13 3x. All solution
should have been removed after last wash.
12. Lid open, air dry the resin in the magnetic stand for 5 mins. No longer than 20 min as
this may inhibit removal of the DNA.
13. Add 100 uL of elution buffer, close lid, and vortex for 2 sec. Place tube at 65C for 5mins.
14. Remove tube, vortex for 2 sec, immediately place in the magnetic stand.
Side note: tube must remain hot until placed in the magnetic stand or yield will decreased.
15. Remove the elution fraction containing DNA to a new tube and store at -20 C.

Amplification of STR’s Using PCR

Side Note: Prepare the reaction mixture- Must be done on ICE TO PREVENT MISPRIMING.
Wear gloves to avoid any contamination and add the following reagents in the order listed
to a sterile 0.2 mL Eppendorf tube.
1. W/purified DNA add the following:
a. Components
 i. Sterile water 16.50 uL
ii. STR 10x buffer 2.50 uL
iii. CTT multiplex primers pair mix 2.50 uL
iv. Tag DNA polymerase 1.0 uL
v. Total 22.5 uL
2. Pipet 2.5 uL of DNA into the tube containing 22.5 uL of PCR mix.
3. Prepare the control reaction (Known template) This is prepared by adding all reagents in
similar manner as previous tube. ONLY difference is that K562 DNA template is added
to the tube.
4. Prepare the negative control this is prepared by adding all reagents in similar manner as
previous tube. ONLY difference is that No DNA is added. Increase amount of DDi-water
added by 2.5 uL.
5. Start Reaction: place all the tube in the Thermal Cycler, and begin the programmed
cycles
a. 94 C for 60 sec, 60 C for 60 sec, 70 C for 90 sec. Repeat 10x
b. 90 C for 60 sec, 60 C for 60 sec, 70 C for 90 sec. Repeat 20x
c. 60 C for 30 min. to finish elongation of all strands
d. Hold at 4 C until next morning. Then store in frig.
6. Preparation of samples for gel electrophoresis: When ready to run gel pipet 1o uL of the
PCR reaction mix into a fresh tube.
7. Add 3 uL of non-denaturing agarose gel loading buffer (0.25% bromophenol blue 40%
sucrose) to the above tube and load your sample onto the gel. Note the lane number of
sample.
8. TA will prepare marker to run alongside samples.
Agarose gel electrophoresis of DNA and amplification products
Side note: you may use agarose gel to confirm the success of PCR reaction quickly before
performing polyacrylamide gel electrophoresis.
1. 2% agarose gel. Add 1.0 g of agarose to 100 mL of 1x TAE buffer. (Dilute from 10x make
1 L for later use)
2. Heat until boiling, then cool to 55 C before pouring into the gel tray
3. While cooling, tape the end of the trays and position the combs. Pour the agarose into
the tray and let cool until opaque. At this point it will be solid.
4. Prepare the samples by mixing the amplified STR’s w/ the dye as specified in the PCR
protocol.
5. Place gel and tray into the electrophoresis gel box pour the running buffer into the tank
over the gel. The buffer should cover the gel but NOT run out of the electrophoresis
unit. Gently remove comb. Load each sample
Side note: make sure it is mixed w/agarose gel loading buffer
6. Load a lane containing size markers. Set voltage at 100 V, and allow the gel to run until
dye is 2/3 of the way down the gel.
7. Add 6 drops of concentrated ethidium bromide soln, mixing in well w/ TEA buffer. Allow
the gel to stain for 30-45 mins
8. Place the gel on a UV transilluminator and photograph gel.
Side note: if you see extra bands in addition to the alleles. DNA heteroduplexes can be
expected when performing nondenaturing agarose gel electrophoresis. The sole purpose of
the agarose gel is to confirm the success of the PCR reaction.