PCR AMPLIFICATION IN RUBBER

Agricultural Biotechnology

MAJOR 5

ALVIN B. FAELDONIA

4-BSA-PBG
INTRODUCTION

The polymerase chain reaction (PCR) is a relatively simple technique that

amplifies a DNA template to produce specific DNA fragments in vitro. Traditional

methods of cloning a DNA sequence into a vector and replicating it in a living cell often

require days or weeks of work, but amplification of DNA sequences by PCR requires

only hours. While most biochemical analyses, including nucleic acid detection with

radioisotopes, require the input of significant amounts of biological material, the PCR

process requires very little. Thus, PCR can achieve more sensitive detection and higher

levels of amplification of specific sequences in less time than previously used methods.

These features make the technique extremely useful, not only in basic research, but

also in commercial uses, including genetic identity testing, forensics, industrial quality

control and in vitro diagnostics. Basic PCR is commonplace in many molecular biology

labs where it is used to amplify DNA fragments and detect DNA or RNA sequences

within a cell or environment. However, PCR has evolved far beyond simple amplification

and detection, and many extensions of the original PCR method have been described.

The polymerase chain reaction (PCR) is a powerful core molecular biology

technique. It is an efficient and rapid in vitro method for enzymatic amplification of

specific DNA or RNA sequences from nucleic acids of various sources. A simple PCR

reaction consists of a set of synthetic oligonucleotide primers that flank the target DNA

sequence, target DNA, a thermostable DNA polymerase and dNTPs. A repetitive series

of cycles involving template denaturation, primer annealing, followed by extension of the

annealed primers, yields tremendous amounts of DNA. Because the strands

synthesized in one cycle serve as a template in the next, a million-fold increase in the

DNA amount is achieved in just 20 cycles.

OBJECTIVES

1. To conduct DNA amplification thru PCR technology

2. To develop skills in doing PCR amplification

3. To identify the bands and banding patterns of 5 rubber varieties.

MATERIALS AND METHODS

The materials used in this laboratory were the following: Horizontal gel

electrophoresis apparatus, D.C. power supply, micropipet with tips, Balance

Microwave, hot plate or burner, Gel casting tray, Gel combs, Electrophoresis tank 250

ml flasks or beakers, vinyl gloves and Distilled water; PCR products, extraction buffer

stock (sorbitol, TrisHCL, EDTA), Lysis buffer stock (TrisHCL, EDTA, NaCl, CTAB),

Sarcosyl stock, Fresh buffer (sodium disulfite, PVP, extraction buffer, lysis buffer,

sarcosyl), vertical rig.

PCR mix (X5) TOTAL

DdH2O 5.1 25.5
10x Buffer 1 5
10 MM dNtP 1 5
50 MM MgCl2 0.8 4
10 MM Primer 0.8 4
Taq 0.3 1.5
DNA (50ng, ul) /10ul/rxn

PRIMER USED: 1 (R20284)

Sample Used:
1. PB217 SUSCEPTIBLE
2. PR107
-
3. PB260 - MODERATE
4. RRIM600
-
TOLERANT
5. GTI - RESISTANT
-
B. Method
S
1. Prepare the PCRUreaction in a properly labeled 96-well PCR plate.
S
2. Dispense 2ul of diluted DNA solution (working DNA stock diluted to 1:20) into each

well.

3. Prepare the cocktail mix in in a 1.5 ml or 2ml micro centrifuge tubes by dispensing the

different components.

4. Mix cocktail thoroughly and aliquot 8ul to each pre-dispensed DNA samples in the

PCR plate.

5. Overlay the mixture with one drop of mineral oil.

6. Cover the PCR plate with adhesive plate and label the PCR plate with your name,

date of preparation, primers used, and entries used in this particular run.

7. Place the plate in a thermal cycler (PCR machine) previously programmed with the

following profile.

8. After the PCR cycles are completed, remove the plate and store in a refrigerator.

DNA AMPLIFICATION

DNA amplification was performed by thermal cycler. The PCR product contains

primers, Taq DNA polymerase, ddH20, PCR buffer, MgCl20, dNTP was mix into
microcentifuge tube. This was place into PCR plate and sealed with adhesive film and

put into the thermal cycler under the PCR conditions it will be (1) initial denaturation-2

minutes at 940C; (2) denaturation-30 seconds at 940C (3) annealing-1 minute at 560C at

1 minute (4) primer extension-1 minute at 720C (5) stage 2 were repeat 29 times (6)

final extension-5 minutes at 720C.

REAGENTS

PCR X5 /r x n

ddH20 5.1 25.5

10 x Buffer 1 5

10mM dNTP 1 5

50mM Mgcl2 .8 4

10mM Primer .8 4

Taq .3 1.5

DNA 50mg, 1ul 10ul/ r x n

Major Steps in PCR

List down the major steps in PCR you have seen the simulation program.
1. Separating the Target DNA—Denaturation

During this stage the cocktail containing the template DNA and all the other core
ingredients is heated to 94-95⁰C.

The high temperature causes the hydrogen bonds between the bases in two strands of
template DNA to break and the two strands to separate.
This results in two single strands of DNA, which will act as templates for the production
of the new strands of DNA.

It is important that the temperature is maintained at this stage for long enough to ensure
that the DNA strands have separated completely.

This usually takes between 15-30 seconds.

2. Binding Primers to the DNA Sequence—Annealing

During this stage the reaction is cooled to 50-65⁰C. This enables the primers to
attach to a specific location on the single-stranded template DNA by way of hydrogen
bonding (the exact temperature depends on the melting temperature of the primers).

Primers are single strands of DNA or RNA sequence that are around 20 to 30
bases in length.

The primers are designed to be complementary in sequence to short sections of

DNA on each end of the sequence to be copied.

Primers serve as the starting point for DNA synthesis. The polymerase enzyme
can only add DNA bases to a double strand of DNA. Only once the primer has bound
can the polymerase enzyme attach and start making the new complementary strand of
DNA from the loose DNA bases.

The two separated strands of DNA are complementary and run in opposite
directions (from one end - the 5’ end – to the other - the 3’ end); as a result, there are
two primers – a forward primer and a reverse primer.

This step usually takes about 10-30 seconds.

3. Making a Copy—Extension

During this final step, the heat is increased to 72⁰C to enable the new DNA to be
made by a special Taq DNA polymerase enzyme which adds DNA bases.
 Taq DNA polymerase is an enzyme taken from the heat-loving bacteria, Thermus
aquaticus. This bacteria normally lives in hot springs so it can tolerate temperatures
above 80⁰C.

The bacteria's DNA polymerase is very stable at high temperatures, which

means it can withstand the temperatures needed to break the strands of DNA apart in
the denaturing stage of PCR.

DNA polymerase from most other organisms would not be able to withstand
these high temperatures, for example, human polymerase works ideally at 37˚C (body
temperature).

72⁰C is the optimum temperature for the Taq polymerase to build the
complementary strand. It attaches to the primer and then adds DNA bases to the single
strand one-by-one in the 5’ to 3’ direction.

The result is a brand new strand of DNA and a double-stranded molecule of

DNA.

The duration of this step depends on the length of DNA sequence being
amplified but usually takes around one minute to copy 1,000 DNA bases (1Kb).

These three processes of thermal cycling are repeated 20-40 times to produce
lots of copies of the DNA sequence of interest.

The new fragments of DNA that are made during PCR also serve as templates to
which the DNA polymerase enzyme can attach and start making DNA.

The result is a huge number of copies of the specific DNA segment produced in a
relatively short period of time.

2.) During the annealing step cycle 1, list down the

a. Sequences of the two primers _5’AGTGCTT3’ and 3’GGCTCGA5’____

b. Color of the polymerase ____Red Orange______
c. How many single strands DNA are formed? ___two strands______
 3.) From cycles 4 to 10, indicate the number of the amplified DNA segments.

Cycle No. No. of the identical molecules

4 16
5 32
6 64
7 128
8 256
9 512
10 1024

4.) For cycle 30, how many identical molecules are amplified?

It has 1073741824 identical molecules amplified.

5.) After 30 cycles of PCR, look and draw the amplified DNA band on gel below.

Results and Discussion

The amplified DNA band on gel showed that big sized fragments stays in above,
while the smaller sized fragments travel to the bottom, and also the longer DNA
fragment above produced greater number of DNA.
REFERENCES:

https://worldwide.promega.com/resources/product-guides-and-selectors/protocols-and-
applications-guide/pcr-amplification/

https://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-
products.html?TablePage=9619242