DNA DIGESTION, POLYMERASE CHAIN REACTION AND GEL ELECTROPHORESIS

NAME: STUDENT ID NO: CLASS: DATE OF EXPERIMENT: NAME OF LECTURER: ZUL FARHAN BIN ZULKIFLI 2010663604 11M2 10th SEPTEMBER 2011 MRS. PUSPARANEE HAKIM

RATIONALE
Everybody in this world is born with different and unique characteristics regarding their physique which originates from their inherited parental genes. We are distinctive by the means of our genetic makeup meaning that our deoxyribose nucleic acid (DNA) are made up of variety of pattern of base sequences. These sequences of bases will give rise to many differences in ourselves such as the colour of our hair, skin and eyes. Since our DNA are microscopic, thus it is invisible to our naked eyes, these protein molecules can only be detected using complex and pricey methods like the ones used in this experiment which includes DNA digestion, polymerase chain reaction and gel electrophoresis. By using these methods, our DNA bases, specific fragment called short tandem repeats (STR), can be detected and compared with other specimens. Usually this is used during forensic investigations to determine the suspect and criminal of a crime scene as DNA is very specific and precise.

OBJECTIVES
The purpose of conducting this experiment is to achieve certain objectives: 1.) Obtain experience of extracting, multiplying and observing DNA segments using specific methods as stated above. 2.) Learn the importance of different buffer solutions used upon DNA molecules. 3.) Train to be precise and careful when carrying steps involving DNA molecules.

INTRODUCTION
Gel Electrophoresis in Forensics Any type of organism can be identified by examination of DNA sequences unique to that species. Identifying individuals within a species is less precise at this time, although when DNA sequencing technologies progress farther, direct comparison of very large DNA segments, and possibly even whole genomes, will become feasible and practical and will allow precise individual identification. To identify individuals, forensic scientists scan 13 DNA regions, or loci, that vary from person to person and use the data to create a DNA profile of that individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of 13 regions.

Figure 1: Result of Gel electrophoresi s under UV light Source: 6

Restriction Enzyme Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA.

from primary data. A 15-question multiple-choice quiz allows you to test your understanding of the material. An additional three questions test your ability to construct restriction maps from DNA fragment size data. The correct restriction maps may be viewed on-screen.

Figure 2: Sticky ends produced after being cut by restriction enzymes Source: 7

Some Examples of DNA Uses for Forensic Identification

Identify potential suspects whose DNA may match evidence left at crime scenes

Exonerate persons wrongly accused of crimes Identify crime and catastrophe victims Establish paternity and other family relationships Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers)

Detect bacteria and other organisms that may pollute air, water, soil, and food
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Identification Using DNA DNA identification can be quite effective if used intelligently. Portions of the DNA sequence that vary the most among humans must be used; also, portions must be large enough to overcome the fact that human mating is not absolutely random. With DNA, the same kind of thinking is used; you can look for matches (based on sequence or on numbers of small repeating units of DNA sequence) at many different locations on the person's genome; one or two (even three) aren't enough to be confident that the suspect is the right one, but thirteen sites are used. A match at all thirteen is rare enough that you (or a prosecutor or a jury) can be very confident ("beyond a reasonable doubt") that the right person is accused. DNA Typing Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists can use these variable regions to generate a DNA profile of an individual, using samples from blood, bone, hair, and other body tissues and products.

Figure 3: Blood is pricked to be extracted as DNA sample Source: 8

In criminal cases, this generally involves obtaining samples from crimescene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions (markers). Scientists find the markers in a DNA sample by designing small pieces of DNA (probes) that will each seek out and bind to a complementary DNA sequence in the sample. A series of probes bound to a DNA sample creates a distinctive pattern for an individual. Forensic scientists compare these DNA profiles to determine whether the suspect's sample matches the evidence sample. A marker by itself usually is not unique to an individual; if, however, two DNA samples are alike at four or five regions, odds are great that the samples are from the same person. If the sample profiles don't match, the person did not contribute the DNA at the crime scene. If the patterns match, the suspect may have

contributed the evidence sample. While there is a chance that someone else has the same DNA profile for a particular probe set, the odds are exceedingly slim. The question is, How small do the odds have to be when conviction of the guilty or acquittal of the innocent lies in the balance? Many judges consider this a matter for a jury to take into consideration along with other evidence in the case. Experts point out that using DNA forensic technology is far superior to eyewitness accounts, where the odds for correct identification are about 50:50. The more probes used in DNA analysis, the greater the odds for a unique pattern and against a coincidental match, but each additional probe adds greatly to the time and expense of testing. Four to six probes are recommended. Testing with several more probes will become routine, observed John Hicks (Alabama State Department of Forensic Services). He predicted that DNA chip technology (in which thousands of short DNA sequences are embedded in a tiny chip) will enable much more rapid, inexpensive analyses using many more probes and raising the odds against coincidental matches.
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Figure 4: DNA probes are used as markers in gel electrophoresis Source: 9

DNA Technology 1.) Restriction Fragment Length Polymorphism (RFLP)

RFLP is a technique for analyzing the variable lengths of DNA fragments that result from digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction endonuclease, cuts DNA at a specific sequence pattern know as a restriction endonuclease recognition site. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments, which are separated using gel electrophoresis. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample. RFLP was one of the first applications of DNA analysis to forensic investigation. With the development of newer, more efficient DNAanalysis techniques, RFLP is not used as much as it once was because it requires relatively large amounts of DNA. In addition, samples
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degraded by environmental factors, such as dirt or mold, do not work well with RFLP. 2.) PCR Analysis Polymerase chain reaction (PCR) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells. With RFLP, DNA samples would have to be about the size of a quarter. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample.

Figure 5: Polymerase Chain Reaction Source: 10

3.) STR Analysis Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. The odds that two individuals will have the same 13-loci DNA profile is about one in a billion.

EXPERIMENTAL HYPOTHESIS
DNA segments can be extracted, multiplied and observed using DNA digestion, polymerase chain reaction and gel electrophoresis respectively.

VARIABLES
1. Manipulated: 2. Responding: - Source of DNA - The detection of DNA fragments on agarose gel under UV light 3. Constant: - Reverse and forward primers used - Restriction enzyme used

MATERIALS & APPARATUS

Materials used are listed below: Prick needles, blood samples, alcohol swab, deionised water, disposable gloves, ethanol, Proteinase K, FIREPOL Master (5l 10x Buffer A, 1l dNTP mix, 0.25 l Taq Polymerase, 36.75l deionised water), agarose powder, TBE (Tris-Borate-EDTA) enzyme Bsu RI (Hae III) , EtBr (Ethidium Bromide), Bromophenol blue, 10kb DNA ladder, 100bp DNA ladder, and buffer solutions (Lysis Buffer, Binding Buffer, Pre-wash Buffer, Wash Buffer, Elution Buffer)

Apparatus used are listed below: Thermomixer, reaction tubes (1.5 ml or 2.0 ml), micropipette, microcentrifuge, micro vortexer, measuring cylinder, Schott bottle and stopwatch.

METHOD
Mammalian blood samples can be stored at room temperature for up to 2 to 3 hours, for short time storage which is up to 24 hours, the samples are stored at -4C. For long term storage, freezing the samples at -20C to -80C are recommended. Multiple thawing and freezing before isolating the DNA should be avoided.

PROCEDURE
A 1.) DNA Extraction Blood is extracted from a subject by pricking one of the fingertips using a pricking needle. Blood that flows out is squeezed into a 1.5ml Receiver Tube at maximum of 200l.

2.)

200l of Lysis Buffer is added into the Receiver Tube and mixed 5 times by pipetting up and down.

3.)

The tube is incubated for 3 minutes at 56C while it is continuously shaken, shortly after that 20 l of Proteinase K is added and vortexed.

4.)

The tube is then incubated again for 5 minutes at 56C, then 200l of Binding Buffer is added and mixed by pipetting up and down for four times or vortexing.

5.)

The lysate is transferred into a RTA Spin Filter Set and is centrifuged for 2 minutes at 12,000 rpm. discarded. The filtrate and Receiver Tube is

6.)

The RTA Spin Filter is transferred into a new RTA Receiver Tube and 500l of Pre-Wash Buffer is added.

7.)

The mixture is centrifuged for 1 minute at 12,000 rpm and again the filtrate and RTA Receiver Tube is discarded.

8.)

The RTA Spin Filter is transferred into a new 2ml RTA Receiver Tube and 700l of Wash Buffer is added.

9.)

The mixture is centrifuged for 1 minute at 13,000 rpm and again teh filtrate and RTA Receiver Tube is discarded. This step is repeated but the RTA Spin Filter is added to the same RTA Receiver Tube and is centrifuged for 4 minutes at 13,000 rpm for ethanol removal.

10.)

The RTA Spin Filter is placed into a 1.5ml Receiver Tube and 100l of Elution Buffer id added. The mixture is incubated for 1 minute at room temperature.

11.)

The mixture is centrifuged for 1 minute at 8,000 rpm and again the RTA Spin Filter is discarded.

12.)

The 1.5ml Receiver Tube is closed and stored at 4C.

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B 1.)

DNA amplification (Polymerase chain reaction) 6 l of DNA solution was obtained from the Spin Filter set and placed into a small Receiver Tube.

2.)

4 l of FIREPOL Master Mix was added to the Receiver Tube as well as 2 l of solution consisted of forward primer and reverse primer.

3.) 4.)

8 l of deionised water was added into the Receiver Tube. The solution was labelled and placed into the thermal cycler machine for 1 hour 30 minutes.

C 1.)

DNA digestion and electrophoresis From a Receiver Tube containing DNA solution, the solution is extracted and placed into a different Receiver Tube containing 16 l of DNA.

2.)

1 l of enzyme Bsu RI (Hae III) was added to the tube. The tube was labelled DNA.

3.)

The Reciever Tube is incubated at room temperature for 1 hour. Gel electrophoresis was set up during the waiting hour.

Procedure for preparing gel electrophoresis

1.)

1.5 g of Agarose gel was weighed by using electronic balance and transferred into a Schott bottle.

2.)

50 ml of TBE (Tris-Borate-EDTA) was prepared by using 5 ml of TBE mixed with 45 ml of deionised water.

3.)

5 l of EtBr (Ethidium Bromide) was added into the bottle and stirred.
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The bottles were placed in a microwave for 2 minutes. Distilled water was added to replace the evaporated water in the bottle.

4.)

A casting tray was prepared and the gel from the bottle was poured into the cast tray. The comb was made sure to be placed inside the tray first before adding the gel.

5.)

The gel was allowed to solidify for 30 minutes. After the gel has solidified, remove the comb from the gel and placed the electrophoresis chamber. the cast tray into

6.)

A tracking dye (Bromophenol blue) was mixed with DNA and PCR samples by pipetting up and down before inserting the wells.

7.)

10l of 10 kb DNA ladder was placed on the first well of the gel and 10 l of 100bp DNA ladder in the last well. The next well was

contained with DNA solution for both Receiver Tubes (DNA and PCR).

8.)

The chamber is closed with cover lid and the devise was attached to the electrophoresis machine via electrodes attached to the chamber.

9.)

The machine was left for 1 hour at least to separate the DNA fragments. After one hour, the electrophoresis machine was switched off.

10.)

The gel was removed from the cast ray and placed into the Gel Documentation System (GDS). Analysis was made using UV light to observe for DNA bands.The picture of the gel was taken and printed.

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RESULT & DATA

1 1 kb DNA ladder 2 3 4 5 100 bp DNA ladder

Table 2: Component of lung volume for Subject A (active stage)

Figure 6: Result of gel electrophoresis under UV light

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DISCUSSION
From the observation of the result above, we could see 4 florescent tabs out of 5 in which 2 of them are very bright while the other 2 in between are dimmer and the last one showed a negative result. We could also see that, despite the transferring of both PCR and DNA digestion samples, only the PCR DNA fragments showed significant results in which they illuminate under UV light. Hence, this explains the function of the PCR method in which the DNA fragments were amplified into many copies while the sample from DNA digestion only has some. PCR had successfully multiplied the number of

identical fragments in Sample 1 and Sample 4 and resulted in high intensity florescence. On the other hand, Sample 2 and 3 showed a slightly dimmer intensity due to less DNA fragments produced during the PCR process. From the DNA ladder above (100bp DNA ladder), we could read from the table that it showed for all respective DNA samples have less than 100 base pairs and less than 28 ng/10l of sample. This means that the DNA samples transferred were identical in terms of base pairs and the weight of the samples. In cases of the DNA digestion, nothing could be observed under UV light as the sample is too light and the fragments produced have lack amount of base pairs to complement the DNA ladder. During DNA extraction, many buffers were used for specific functions to be forwarded to digestion and PCR. The Lysis Buffer and Proteinase K are both used to regulate a suitable and ideal environment so that the DNA samples can be cut using the restriction enzyme during DNA digestion to produce specific DNA sequences called the short tandem repeats (STR). Besides that, the Binding Buffer was essential so that the primers (forward and reverse) can anneal to the DNA fragment (or called STR for short tandem repeats), thus they must be complementary to one another. In order to ensure htat the DNA samples were clean any has no residual contaminations, the tubes used were inserted the Pre-Wash Buffer and the Wash Buffer followed by centrifugation so that the entire residue can be settled down inside the Receiver Tubes and consequently discarded. Lastly the Elution Buffer was
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applied as the final washing buffer so that it is ready for any downstream applications like the gel electrophoresis. By this mean also, the DNA samples can be stored at 4C to 8C up to 2 months and 5 years if stored at -20C. During DNA digestion, the restriction enzyme was introduced namely the Bsu RI (Hae III) enzyme. Haemophilus aegyptius is the genomic source for HaeIII. This enzyme works at 37C and cuts the site between G and C bases to STR. The incubation at 37C for 1 hour was necessary to ensure that all GC segments are separated. Unlike in Sample 5, where there were no results produced in the gel electrophoresis. This could mean that Sample 5s DNA has the absence of G-C segment in his DNA strand. During the PCR, 4 types of phases were undergone for each samples. Each phases were repeated about 30 times so that sufficient amount of STRs are replicated to show results in gel electrophoresis. This was successful for Sample 1 and 4, thus showing florosence. The first and second phase was practically the same thing which the DNA was heated to 94C to denature the DNA strands by breaking the hydrogen bonds that hold the double strand s together. After that the third phase was to anneal the forward and reverse primer to the STR in which the mixture was heated at 60C. The forward primer used consists of 5GAA ACT GGC CTC CAA AGA CTG CCC 3 while the reverse primer is 5 GTC TTG GAG ATG CAC GTG CCC CTT GC 3. These primers are complementary to the sticky ends of the STR produced during the DNA digestion. Only the STRs containing the ends of a C or G base can be annealed and elongated via nucleotide condensation. This step is the fourth phase, where the samples are heated at 72C for 5 minute. As a result, many copies of the STR is produced and ready for gel electrophoresis. In Sample 2 and 3, the amount of STR present became the limiting factor to produce sufficient amount of copies, unlike for Sample 1 and 4 where their initial amount of STR are plenty, thus being replicated at a faster rate. In gel electrophoresis some reagents are necessary for specific functions. The EtBr will bind with STR and it will emit florescence under UV light. The TBE solution (Tris-Borate-Edta) provides an ionic solution that allows current to pass through the water.
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The Tris (and the fact that the buffer is basic) part is useful to keeping the DNA deprotonated and in solution.

EDTA protects nucleic acids from degradation. It removes certain metal ions from enzymes that would destroy the nucleic acid, thus inactivating the enzymes. The advantages of putting this method into practical is that DNA can be extracted quickly and more conveniently. Besides that, the contaminants such as proteins and nucleases, which may interfere with PCR reactions, are completely removed, thus improving the efficiency and reproductibility of PCR. Other than that, damages to DNA can be reduced by avoiding any precipitation and the usage of organic solvents. Lastly, the isolated DNA is ready for use in various applications as it is contained and stored in a safe and convenient manner.

VALIDITY AND RELIABILITY

This experiment has portrayed valid sets of results because the agarose gel showed a positive outcome in which 4 out of 5 samples showed florosence under UV light. This result prove that the reagents and the DNA samples that we used are mostly extracted and mixed properly despite in absence of the Binding Buffer during the DNA digestion. Nevertheless the environment suited the DNA samples to bind with the primers and undergo nucleotide condensation during PCR. Thus, amplifying the samples for gel electrophoresis. Personally, I believe that our data are reliable because we did a set of 5 samples per group. This means that our data is confirmed collectively by all samples except for 1. The data for our group also showed the same results by the DNA ladder, which confirms that the enzymes and primers used were reliable and is the same for all DNA samples.

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SOURCE OF ERRORS
Along the experiment, there were few sources of error that may possibly affect our data in the Gel Documentation System under UV light. Firstly, there is a low yield of DNA or low levels of purity of DNA. This could be caused when buffers or other reagents may have been exposed to condition that reduce their effectiveness. Besides that, the reagents and samples used may not have been completely mixed. In addition, the lysis process may also be incomplete. In other cases, there could be an incomplete, or no restriction, enzyme cleavage of isolated DNA. Furthermore, the DNA tissue samples may have been degraded during the experiment. Lastly, there may be white

precipitate in some buffer solution like the Lysis Buffer or Binding Buffer after prolonged storage at low temperature. These errors can be avoided as

discussed in the modification part of the report later on.

SAFETY MEASURES
Safety measures are vital to protect us from any injuries or casualties when conducting the experiment. Firstly, the solvent EtBr is proven to be highly carcinogenic even in minute amount, thus contact with this solvent must be avoided by wearing disposable gloves. The marker dyes are highly

concentrated blue in colour and lab coats are advised to be worn to avoid any stains to appear on experimenters clothes. Furthermore, the apparatus are handled in a careful manner especially the Schott bottles when it is instantly heated via the microwave because it could be very hot. Cooling the bottles before handle is necessary to avoid any burns.

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LIMITATIONS
Limitations are the factors that lack in the experiment which in turn may lead to difficulties and delay to carry the investigation collectively. One of the limitations that we had encountered was the lack of mastery in using the apparatus provided especially when using the micropipette to transfer the PCR and DNA digestion fragments into the solidified agarose gel. Besides that, the apparatus provided are lacking, like the micropipettes which forced us to make queues in order to obtain buffer solutions before being centrifuged. This had taken some of our time while we could have finished earlier. Moreover, the difficulty of squeezing the blood from the fingertips also had consumed a major part of our time during the first day of experiment. While some of us have low blood pressure or fast clotting period, this problem had led us to take longer time to complete the experiment.

MODIFICATIONS
This experiment requires some modifications to allow the results to become more accurate and significant. Firstly, correct technique of extracting blood should be explained clearly to the experimenters so that more time can be saved to do further steps instead of hurting the fingertips to create openings for blood efflux. Other than that, the correct usage of apparatus such as the micropipette, when inserting the DNA fragments into the agarose gel, should be taught practically so that the experimenters can avoid any mistakes like poking and consequently leaking the agarose gel. Moreover, the quantity of DNA samples per person can be increased so that if a mistake happens, the troubled step can be repaired by using the extra supply of DNA samples. The sources of error can be neglected by some steps. Firstly, we must ensure that the reagents used like the buffer solutions are stored at room temperature at all times upon arrival, and that all reagent bottles are closed tightly after use to preserve pH and avoid contamination. Moreover, during the mixing of DNA samples with the reagents, the clarity of colour of the sample
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changes from turbid to clear, indicating that protein digestion has occurred. For example, lysis process takes about 1 to 3 hours and can be speed up using shaking water bath. Besides that, to avoid any degradation of sample tissues, they should be frozen at -20C immediately after harvesting and remain so until the lysis procedure starts. Lastly, the buffers solutions are kept in a uniform state where it does not contain any impurities inside them by incubating them at 60C. The precipitate will dissolve at high temperature and will not alter the yield and quality if the purified nucleic acids.

FURTHER WORKS
Some further works are necessary in this investigation regarding the different base sequences that is integrated in our DNA in order to dig deeper about the correlation it has with other specimens like other mammals. One of the ways this can be experimental is by using different sets of reverse primers, forward primers and restriction enzymes so that different DNA segments can be extracted, amplified and compared. Other than that the source of DNA can also be altered by taking samples from the cheek cells or hair. Other than that, the DNA of identical twins and fraternal twins can be investigated by using gel electrophoresis to analyse the degree of similarity that they share as twins.

CONCLUSION
DNA segments can be extracted, multiplied and observed using DNA digestion, polymerase chain reaction and gel electrophoresis respectively. Hypothesis is accepted. Objectives were achieved: 1.) Obtain experience of extracting, multiplying and observing DNA segments using specific methods as stated above. 2.) Learn the importance of different buffer solutions used in each method . 3.) Train to be precise and careful when carrying steps of different methods
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