UNIVERSITI TEKNOLOGI MARAFACULTY OF CHEMICAL ENGINEERING

GENETICS LABORATORY(CBE561)

NAME / ID : 1) SITI AISHAH BINTI SULAIMAN (2010653746)

2) NUR ATHIKA SHAKIRA BT JAIS (2010285582)

3) MARINA BT YUSOFF (2010672752)

4) NOR FARAHIDA BT ABU BAKAR (2010811294)

5) ADIBAH DAYANA BT AHMAD (2010837536)

EXPERIMENT : The isolation of genetic material by using PCR

DATE PERFORMED : 6th June 2012

PROG/CODE : Bachelor of Engineering (Hons.) in Chemical & Bioprocess Engineering / EH222 4B

SUBMIT TO : Dr. Tan Huey Ling ( )/ Mdm. Nurul Huda Mohamed Safri( )

NO Title Allocated Marks % Marks %

1 Abstract 52 Introduction 53 Aims 54 Theory 55 Apparatus 56 Procedure 107 Results 108 Calculations 109 Discussion 2010 Conclusion 1011 Recommendations 512 References 513 Appendix 5

TOTAL 100

Remarks:

Checked by;

Date :

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TABLE OF CONTENTS

No. Contents Page

1. Abstract 3

2. Introduction 4

3. Objective 5

4. Theory 5-6

5. Apparatus 7

6. Experimental procedures 8-9

7. Results 10

8. Calculations 11

9. Discussion 12-13

10. Conclusion 14

11. Recommendations 15

12. References 15

13. Appendices 16

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ABSTRACT

Polymerase chain reaction (PCR) allows researchers to amplify DNA in a test tube. This

process uses an enzyme derived from heat-resistant bacteria. The steps of PCR are driven by

changes in temperature.

Polymerase chain reaction, or PCR, uses repeated cycles of heating and cooling to

make many copies of a specific region of DNA. First, the temperature is raised to near

boiling, causing the double-stranded DNA to separate, or denature, into single strands. When

the temperature is decreased, short DNA sequences known as primers bind, or anneal, to

complementary matches on the target DNA sequence. The primers bracket the target sequence

to be copied. At a slightly higher temperature, the enzyme Taq polymerase, shown here in

blue, binds to the primed sequences and adds nucleotides to extend the second strand. This

completes the first cycle. In subsequent cycles, the process of denaturing, annealing and

extending are repeated to make additional DNA copies. After three cycles, the target sequence

defined by the primers begins to accumulate. After 30 cycles, as many as a billion copies of

the target sequence are produced from a single starting molecule.

The four micro test tubes was obtained and labelled as yellow tubes for lambda DNA,

violet tube for Pstl lambda digest, green tube for EcoR1 lambda digest and orange tube for

Hind iii lambda digest. The components was mixed by gently flicking the tube with finger

been tapping gently on the table to collect liquid to the tube bottom. The tube in a centrifuge

is pulse-spin to collect all the liquid to the bottom, or gently been trapped on the benchtop and

then the four micro test tubes was being incubated. After that, the agarose gel electrophoresis

was prepared. Next, 10ul of each sample is loaded into separate wells in the gel chamber and

the liquid is placed carefully on the electrophoresis chamber. When the electrophoresis is

completed, the power is turned off and the top of the chamber is removed. The gel and tray is

been removed carefully from the gel box. After that, the result that obtained was analysed and

if the band does not appear clearly then the gel was stained and the gel was being analyse

once again.

Based on the result, it shows that EcoR1 and Hind III had the most similar band size.

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INTRODUCTION

DNA cloning in cells remains the best method for preparing large quantities of a

particular gene or other DNA sequence. However, when the source of DNA is impure, the

polymerase chain reaction or PCR is quicker and more selective. In this technique, any

specific target segment with one or many DNA molecules can be quickly amplified in a test

tube. With automation, PCR can make billions of copies of target segment of DNA in few

hours significantly faster than the days it would take to obtain the same number of copies by a

screening DNA library for a clone for a desired gene and it replicate within a host cells.

In the PCR procedure, a three step cycle brings about a chain reaction that produces an

exponentially growing population of identical DNA molecules. During each cycle, the

reaction mixture is heated to denature the DNA strands and cooled to allow the annealing

(hydrogen bonding) of short, single stranded DNA primers complementary to sequence an

opposite strands at each end of the target sequence; finally, a heat-stable DNA polymerase

extends the primers in 5’-3’ direction. If a strand DNA polymerase were used, the protein

would be denatured along with the DNA during the first step and would have to be replaced

after each cycle. The key to automating PCR was the of an unusual heat-stable DNA

polymerase, first isolated from the cells of a bacterial species living in a hot spring, that could

withstand the heat at the start of each cycle. Only minute amounts of DNA need to be present

in a starting material, and this DNA can be partially degraded state, as long as few molecules

contain the complete target sequence. By the end of the third cycle, one-fourth of the

molecules are identical to the target segment, with both strands the appropriate length. With

each successive cycle, the number of target segment molecules of the correct length doubles,

soon greatly outnumbering all other DNA molecules in the reaction.

Usually, gel electrophoresis is a widely used in the analysis of nucleic acids and

proteins. Agarose gel electrophoresis is used for the preparation and analysis of DNA. It is

a procedure that separates molecules on the basis of their rate of movement through a gel

under the influence of an electrical field. DNA is negatively charged and when placed in an

electrical field, DNA will migrate towards the positive pole (anode). An agarose gel is used to

slow the movement of DNA and separate by size. Within an agarose gel, linear DNA migrates

inversely proportional to the log10 of their molecular weight.

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OBJECTIVE

To setting up the Polymerase Chain Reactions

To run electrophoresis of Polymerase Chain Reactions product

To answer question based on Polymerase Chain Reactions

THEORY

The polymerase chain reaction (PCR) is a molecular genetics technique for making multiple

copies of a gene, and is also part of the gene sequencing process. Gene copies are made using

a sample of DNA and the technology is good enough to make multiple copies from one single

copy of the gene found in the sample. PCR amplification of a gene to make millions of copies,

allows for detection and identification of gene sequences using visual techniques based on

size and charge of the piece of DNA. Under controlled conditions, small segments of DNA

are generated by enzymes known as DNA polymerases that add complimentary

deoxynucleotides (dNTPs) to a piece of DNA known as the template. A smaller piece of

DNA, called primers is used as a starting point for the polymerase. Primers are small man-

made pieces of DNA (oligomers), usually between 15 and 30 nucleotides long. They are made

by knowing or guessing short DNA sequences at the very ends of the gene being amplified.

During PCR, the DNA being sequenced is heated and the double strands separate. Upon

cooling, the primers bind to the template and create a place for the polymerase to begin. PCR

was made possible by the discovery of thermophiles and thermophilic polymerase enzymes.

This is an enzyme that maintains structural integrity and functionality after heating at high

temperatures.

As the name implies, it is a chain reaction, a small fragment of the DNA section of interest

needs to be identified which serves as the template for producing the primers that initiate the

reaction. One DNA molecule is used to produce two copies, four, eight and so forth. This

continuous doubling is accomplished by specific proteins known as polymerases. Polymerase

is an enzymes that are able to string together individual DNA building blocks to form long

molecular strands. Polymerases require a supply of DNA building blocks which is the

nucleotides consisting of the four bases adenine (A), thymine (T), cytosine (C) and guanine

(G). They also need a small fragment of DNA, known as the primer, to which they attach the

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building blocks as well as a longer DNA molecule to serve as a template for constructing the

new strand. If these factors are supplied, this enzyme will construct exact copies of the

templates. The polymerase chain reaction serves to copy DNA. It uses repeated cycles, each

of which consists of three steps:

4.1: Denaturing

The reaction solution containing DNA molecules , polymerases, primers (serve as starting

DNA) and nucleotides (which are attached to the primers) is heated to 95°C. This causes the

two complementary strands to separate, a process known as denaturing or melting.

4.2: Annealing

Lowering the temperature to 55°C causes the primers to bind to the DNA, a process known

as hybridization or annealing. The resulting bonds are stable only if the primer and DNA

segment are complementary. The polymerases then begin to attach additional complementary

nucleotides at these sites, thus strengthening the bonding between the primers and the DNA.

4.3: Extension

The temperature is again increased to 72°C. This is the ideal working temperature for the

polymerases used, which add further nucleotides to the developing DNA strand. At the same

time, any loose bonds that have formed between the primers and DNA segments that are not

fully complementary are broken. Each time these three steps are repeated the number of

copied DNA molecules doubles. After twenty cycles, about a million molecules are cloned

from a single segment of double stranded DNA.

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APPARATUS

PCR tubes

Cap less micro centrifuge tube

Pipette

Pipette tip

Ice

Thermal cycle

Pulse spin

Electrophoresis

Gel box

MATERIALS

Template DNA

Blue MMP

Orange G Loading Dye

Agarose gel

Ice

Allele ladder

METHOD

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5.1: Setting up the PCR Reactions

1. Five PCR tubes are labelled as CS, A, B, C and D by including our groups members. The PCR tubes are placed into a cap less micro centrifuge tube on ice.

2. 20μl of appropriate template DNA is transferred correctly into labelled tube by follows table below.

Label PCR tubes Add DNA template Add Master mix + primers

CS + your initials 20μl Crime Scene DNA 20μl MMP(blue)

A+ your initials 20μl Suspect A DNA 20μl MMP(blue)

B+ your initials 20μl Suspect B DNA 20μl MMP(blue)

C+ your initials 20μl Suspect C DNA 20μl MMP(blue)

D+ your initials 20μl Suspect D DNA 20μl MMP(blue)

3. 20μl ot MMP (master mix + primers) are transfer into each of 5 PCR tubes containing template DNA. Pipette up and down to mix. Cap each tube after adding blue MMP.

4. Capped PCR tubes are placed in their adaptors on ice.

5. Tubes are placed in the thermal cycler when instructed to do so. Thermal cycle for PCR is programmed.

5.2: Electrophoresis of PCR Products

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1. Gel electrophoresis equipment is set up as instructed.

2. Five PCR tubes are obtained from the previous lesson. The PCR tube is placed in cap less tube and pulse-spin in a balanced micro centrifuge for a few seconds to collect all liquid to the bottom of the tubes.

3. 10 μl of Orange G loading dye (from the tube labelled LD) is transfer into each PCR tubes. Pipet up and down to mix and pulse spin to collect liquid in the bottom of the tubes.

4. An agarose gel is placed in the gel electrophoresis apparatus. The well of the agarose gel is checked near the black (negative) electrode and the base of the gel is near the red (positive) electrode.

5. The electrophoresis chamber is filled with enough 1XTAE buffers to cover the gel. This required about 275 ml of 1 X TAE buffer.

6. Load 20 μl of the sample by using a clean tip for each sample into six wells of the gel in the following order:

Lane Sample Load volume

1 Allele Ladder 20 μl

2 Crime Scene 20 μl

3 Suspect A 20 μl

4 Suspect B 20 μl

5 Suspect C 20 μl

6 Suspect D 20 μl

7. The lid on the gel box is secure. The lid is attached to the base in only one orientation: red to red and black to black. The electrical lead is connected to power supply.

8. The power supply and electrophoresis sample is turn on at 100 V for 30 minutes.

9. Fast Blast DNA stain is used to stain the sample.

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RESULT

Ladder(bp)

Crime scene (bp)

Suspect A(bp)

Suspect B(bp)

Suspect C(bp)

Suspect D (bp)

15000

1000 1000 1000

700 700 700

500 500400300 300 300 300200 200 200100

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CALCULATIONS

Calculation in preparation of agarose gel

1g = 1% agarose

1g agarose = 100ml of buffer

4g agarose = 400ml of buffer

8 agarose gel = 400ml of buffer

M1V1 = M2V2

(500)(V1) = (1)(3)

V = 0.06L

3 - 0.06 = 2.94 L H20

50 X TAE H2O

(0.06 L TAE) ÷3 2.94 L H2O÷3

0.02 L TAE 0.98 L H2O

20ml 9.80ml

Master mix primer (MMP) stock for 8 groups

= 1000 µL of Master Mix + 20 µL crime scene investigator primer

Mix well & pulse-spin to bring contents to the bottom of the tube

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DISCUSSION

Crime is an action or omission that constitutes an offense that may be prosecuted by the state

and is punishable by law. In other words, crime is an action or an instance of negligence that

is deemed injurious to the public welfare or morals or to the interests of the state and that is

legally prohibited. Crime can occurs in various forms and to anyone, not limited to gender,

age or place. Nowadays, rape, murderer is not a new thing to our ears. The lack of witnesses,

lack of evidences, and lack of brave people to come forward and give witnesses to a crime

scene led to a new discovery of technology that can uphold justice.

For sure, the criminals would not leave any evidence at the crime scene, the samples

obtained from the crime scene found usually too small and almost impossible to be analyst.

Thanks for technologies that make it easier for human to uphold justice. As for the forensic

lab analysis, non-coding DNA is used in a method of distinguishing between individuals by

analysing patterns in their DNA. Non-coding DNA describes components of an organism's

DNA sequences that do not encode for protein sequences which not transcribed into

messenger RNA to code for proteins.

In this study, firstly PCR technique is used. Basically, PCR act to amplify the DNA

segment. This is done by denaturing the DNA sample, having primers anneal then extension

of the annealed primers by polymerase and the process being repeated multiple times.

Secondly, we used gel electrophoresis. When putting DNA sample above the gel, different

size of DNA will move at different speed and different length of pathway. DNA is negatively

charged particle and will move towards the positively charged pole above the agarose gel.

This allow the DNA to separate by size, as the size is lighter, it will accelerates faster towards

the opposite pole, thus comparisons can be made to the ladder and the crime scene DNA.

With the aid of PCR or polymerase chain reaction method, any segment or target

sequence within the DNA sample can be copied many times (amplified). PCR requires

double-stranded DNA containing the target sequence, a heat-resistance polymerase, all four

nucleotides, and two 15 to 20 nucleotide DNA strands that serve as primers. One primer is

complementary to one end of the target sequence on one strand; second primer is completely

to the other end of the sequence on the other strand.

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Theoretically, the suspected criminal is individual C. At the end of the experiment we

realized that some error occur during conducting our experiment as the results deviate from

the theory There are two bands that possess the same genotype which is 7-3. The result shows

that the suspect individuals are suspect’s B and D.

Based on our analysis, individual B is suspected to be a crime scene sample whereas

individual D is suspected to be the criminals (suspect C). This is because they possess the

same genotype which is 7-3, the other three suspects are genotype of 5-2, 10-3, and 10-2

respectively. The DNA of each of them had two segments that each migrated displaying that

they were in the same size, thus suspect D which is actually C was proven to be at the crime

scene by the PCR technique. There are some errors that will be discussed further in next

paragraph.

During conducting this experiment, it is compulsory for student to label the sample for

each test tube, the macro and the micro one. Our group forgot to label the small test tube

which results in wrong finding. At the end of the experiment only we realize that we have

mixed the sample without labelled them correctly. This finally leads to confusing of sample

crime scene DNA. Another important thing that results in error our experiments are lack of

pipette. Students have to share the same pipette tip for different types of chemical used. The

sharing of pipette tip during pipetting those chemical reagents into the tube results in mixing

of various samples. One pipette tip should be use in transferring of only one chemical.

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CONCLUSION

Based on the experiment, PCR or Polymerase Chain Reaction can be used to amplify a

specific region of the DNA, when DNA samples from spots of blood or hair roots do not give

enough material for forensic analysis. The primers and probes used act as a genetic marker to

differentiate individuals.

As a conclusion the aim of this experiment , (to understand in details the useful of Polymerase

Chain Reaction (PCR) and how to conduct the procedures, manage to find the real suspect by

comparing the sample DNA and the suspect DNA using the gel electrophoresis and knowing

what are the uses of restriction endonucleases in DNA sequence) are not completely achieved

as the third objectives that is manage to find the real suspect is failed. This is due to the some

error occur during labelling the suspect’s sample.

Based on this forensic analysis and discussions above, the genotype of crime scene is 7-3. The 4

suspects have genotype of 10-3, 5-2, 7-3 and 10-2.Suspect C genome matches the crime scene

sample genome. Therefore, another suspect such as A,B,D are considered to be excluded from

the crime. This is because, based on the PCR results, the DNA for suspects A,B and D does

not match to the crime sample genome. To conclude, suspect’s C was proven as a criminal.

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RECOMMENDATIONS

There are some recommendations to the viewers in the future regarding this experiment so as

to conduct this study well:

• It is advisable for the student to not over stain the agarose gel as it will takes a longer time to

bring the gel into neutral color. Thus, the longer time taken to observe the movement of bands

above the gel.

• Make sure that the solutions are brought to centrifugation so as to allow it to mix well.

• Preparation of excess stock for DNA primer is suggested instead of assuming it to be enough

throughout this study. This excess stock can be used if there is a mistake occurs involving so

much consuming of stock solution or spillage.

• If the tip of pipette is not enough, it is recommended to wash them with distilled water

properly for other uses. This can prevent mixing of various chemicals used.

• Make sure that the pipette used is accurate because in this experiment.For a better result,

make sure that the gel is slowly poured into its container so as to avoid formation of bubbles.

Bubbles can cause error in reading the result. It can be removed with a pipette tip.

REFERENCE

1. Bustamante, I T, Mata, FS, et al (2007) Application of chemometric tools for

automatic classification and profile extraction of DNA samples in forensic tasks,

Analytical Chimica Acta, 595, pg 43-50

2. Campbell N. Reece J & Meyers N. Biology, 7th Edition, pg 402-406. Pearson

Education, Australia 2006.

3. Lodish, H. Berk, A., et al (2004). Molecular Cell Biology, pg 87-89, W H Freeman

andcompany, New York.

4. Laboratory Studies in Applied Genetics (21 April 2009). Methods manual. Retrieved

on 9th November 2011 from

http://www.jblearning.com/samples/076371075X/Wheelis_CH03_025%20copy.pdf

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APPENDIX

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