BIOLOGY LAB REPORT TITLE : DNA (EXTRACTION, AMPLIFICATION AND DIGESTION) AND ELECTROPHORESIS PREPARED BY I/C NUMBER STUDENT

ID GROUP LECTURERS NAME PRACTICAL DATE SUBMISSION DATE : : : : : : :

Abstract In this experiment, DNA from each student was isolated from extracted blood sample using Lysis Buffer HL, Binding Buffer HL, Pre-Wash Buffer, Wash Buffer and Elution Buffer D. After that, the DNA was used to amplify into many copies via Polymerase Chain Reaction where it undergoes several steps in different temperatures to produce massive amount of identical DNA. This is continued with digesting the DNA by using digestion buffer and Hind III enzyme. The final step in this experiment was to do DNA Gel Electrophoresis where the DNA is mounted into the agarose agar well, together with the markers and run at 100V by connecting to circuit for about 1 hour. The image of DNA band later revealed under UV imager. From this experiment, one can realize that each and every organism on this earth has unique band of DNA which differ from each other and the steps to extract, isolate, digest, amplify DNA and finding DNA Band.

Introduction 1. DNA

Figure 1 : DNA Molecule(1) DNA is a long polymer made from repeating units called nucleotides. As first discovered by James D. Watson and Francis Crick, the structure of DNA of all species comprises two helical chains each coiled round the same axis, and each with 3.4 nm and a radius of 1.0 nm. DNA exists as a pair of molecules that are held tightly together in eukaryotic. These two long strands entwine in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a nucleobase, linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide.

Polymers comprising multiple linked nucleotides (as in DNA) are called a polynucleotide. The backbone of the DNA strand is made from alternating phosphate and sugar residues. The sugar in DNA is a pentose sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among the aromatic. (2)

Figure 2: DNA Replication (2)

Figure 3: Transcription and Translation in Eukaryotic(2)

2. DNA Fingerprinting The steps in DNA fingerprinting are illustrated below:

Figure 4 : Electrophoresis(3)

Figure 5 : Southern Blotting(4)

Southern Blotting is used as a method to identify genes related to a specific function. They also can use the results to compare genes of the same species that have different variations. Southern Blotting help to focus on a gene molecule and are used in evolution in development studying between modern and prehistoric genes. (2) If the amount of DNA sample is too small, PCR reaction, known as polymerase chain reaction can be applied. (2)

Figure 6: PCR Reaction(5)

The most standard form of set up seen is: Denature: 95C for 2 min cycle beginsDenature: 95C for 15 seconds Annealing Temperature: 50-60C for 15 seconds Extension: 72C for 15 seconds cycle ends Extension: 72 for 10 minutes Store PCR product in 4C

Denature Step

Objective To extract DNA from human blood sample, amplify the DNA using PCR reaction, digest DNA into fragments using restriction enzymes and separate and identify DNA fragments by gel electrophoresis. Problem Statement Can DNA be isolated from human blood, amplified, digested and carried out on electrophoresis to produce a DNA profile? Hypothesis DNA can be isolated from blood samples, amplified and digested and electrophoresis can be carried out on DNA to produce a DNA profile. Apparatus Eppendorf tube, micropipette, vortex machine, incubator, RTA Spin Filter Set, centrifuge, 1.5ml RTA Receiver Tube, incubator, gloves, paper tissues, Invisorb Spin Blood Mini Kit Materials Human blood sample, Elution Buffer D, Lysis buffer HL, Proteinase K, Binding Buffer HL, pre-wash buffer, wash buffer, forward primers, reverse primer, Buffer A, dNTP mix, Taq polymerase, deionized water, digestion buffer, enzyme (Hind III), distilled water, agarose gel, ethidium bromide,

Procedure : DNA Isolation from Human Whole Blood A. Isolation of DNA from blood. (DNA extraction) 1. 200l blood was transferred into a 1.5ml Receiver Tube. 2. 200l of Lysis Buffer HL and the mixture was mixed about 5 times using micropipette by pipetting up and down. 3. The mixture was incubated for 3 minutes at 56C while continuously shaking. 4. 20l of Proteinase K was added a nd vortexed shortly. The receiver tube containing the mixture was then incubated for 5 minutes at 56C while continuously shaking. 5. 200 l of Binding Buffer HL was added and mixed by pipetting the solution up and down about four times. The mixture was then vortexed using the vortex mixer. 6. The lysate is then transferred to a RTA Spin Filter and then it was centrifuge for 2 minutes at (12 rpm). The filtrate was discarded and the RTA Spin Filer was placed into a new 2.0 ml RTA Receiver Tube. 7. 500 l of Pre-Wash Buffer was added to the RTA Spin Filter. The spin filter was closed and centrifuged for 1 minute at 12rpm. The filtrate was discarded again and the RTA Spin Filer was placed into a new 2.0 ml RTA Receiver Tube. 8. 700 l of Wash Buffer was added and was left to centrifuge for 1 minute at 12rpm. Again, the filtrate and RTA Receiver Tube were discarded. 9. Step 8 was repeated but the RTA Spin Filter was added to the same RTA Receiver Tube. It was then centrifuged for 4 minutes at 12rpm to remove ethanol. 10. The RTA Spin Filter was placed into a 1.5ml Receiver Tube. About 200 l of preheated (56C) Elution Buffer D was added. 11. The mixture was incubated for 1 minute at room temperature and centrifuged for 1 minute at 9.5rpm. the RTA Spin Filter was discarded. 12. The 1.5ml Receiver Tube was closed and stored at 4C.

B.

DNA Amplification using Polymerase Chain Reaction (PCR) 1. 1l of forward primer and 1l of reverse primer was measured with a 20l micropipette and poured into a PCR tube. 2. 5l of DNA was measured with a micropipette and poured into the clean PCR tube. Next, 9ml of distilled water was added. About 43l pre-mix solution that contains 5l 10x Buffer A, 1l dNTP mix, 0.25 l Taq polymerase and 36.75l deionized water was added. The tube was then labelled. 3. 59l of distilled water was then added into another PCR tube and labelle d as control. This was the negative control for the reaction. 4. Both the tubes were then placed into the Thermocycler for amplification. 5. PCR was performed at 94C for 20 minutes for denaturation to occur, 64 C for 1 minute for annealing to occur and followed by 72 C for 1 minute for extension to occur. 6. The PCR cycle was then repeated for 30 cycles and let to stand at 4C. All this was done within 1 hour and 30 minutes. 7. Finally, the DNA was stored at -20 C to keep it intact.

Recommended PCR cycles: Operation Initial Denaturation Initial Denaturation Annealing Elongation Final Elongation Temperature / C 95 95 54-66 72 72 Time 3-5 mins 20-40 sec 30-60 sec 40sec - 4 mins 5-10 mins Cycles 25-30 25-30 25-30 -

C. DNA digestion

Restriction Enzyme Digestion 1. A 6l of DNA after undergoing PCR amplification is added with 2l digestion buffer (buffer R) using micropipette. 2. It is further added with 11l of water and 1 unit enzyme of Hind III . 3. The mixture is mixed by tapping gently the tube. 4. It is then microcentrifuged for 2 seconds and 1200rpm. 5. After centrifuged, it is incubated for 1 hour at 37C and centrifuged for 5 seconds at 1200rpm.

D.

DNA Gel Electrophoresis

1. TBE is prepared by mixing 5l of tBE and 45ml of distilled water. 2. 0.75g of agarose powder is weighed using weighing machine. 3. It is then mixed with TBE solution and Ethidium Bromide was added. 4. 0.75g of agarose was measured by using electronic balance and then dissolved in 50 ml of 1 TBE in Schott bottle. 5. The gel solution was heated in a microwave oven for 3 minutes until it became clear. 6. Water was added to replace the evaporated water and to level the volume of solution back to 50ml again. 7. 5l of ethidium bromide was added to the solution and the bottle was swirled gently. 8. The molten agarose gel was then left to cool to about 60C and then poured into a casting tray, with the comb placing in the tray. 9. The agarose gel was left to solidify for about 30 minutes. 10. The electrophoresis tank was filled with 500ml 1 Tris-Borate EDTA (TBE) solution. The comb was removed and the gel was submerged in the solution. 11. 10l of marker (1 kb DNA ladder) was loaded into the first well while another 10l of marker (100bp DNA ladder) was loaded into the last well formed on the agarose gel. 12. 20l of DNA samples from the DNA digestion were loaded into the second well, followed by loading 20l of PCR products into the third well and lastly 20l of control solution into the fourth well. 13. The circuit was connected and the gel electrophoresis was run at 100 Volts for about 70 minutes. 14. The circuit was disconnected and the fluid on the solidified agar was poured away. 15. The agar was placed into a UV imager to reveal the bands formed by the DNA fragments. 16. The images formed were scanned into the computer and printed out to be analysed. 17. The agarose was disposed properly as it is a biohazard waste.

Safety precaution In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when conducting an experiment in the lab at all times to protect the skin and clothing from spillage of any chemical substance or blood. Wear hand gloves when handling the agarose gel with ethium bromide because it is slightly carcinogenic. Hands need to be thoroughly washed before and after taking blood. This is to avoid ourselves from getting infected from any of the microorganism. Furthermore, the glassware such as Schott bottle should be handled with full care because they are fragile. The apparatus such as Invisorb Spin Blood Mini Kit is also sterilized to prevent

infection. After using all samples and apparatus at the end of experiment, they should be discarded properly and returned back to their places to avoid injuries and unnecessary accidents that may result fatal results. Liquid waste must be considered infections and discarded according to local safety regulations.

Risk Assessment Invisorb Spin Blood Mini Kit must not be shared to reduce the contamination as well as prevent the transfer of diseases through the blood The tip of the micropipette has to be changed each time between liquid transfers to avoid cross contaminations of liquid the components of different kit were taken care not to combine to prevent contamination. All sterile apparatus such as Invisorb Spin Blood Mini Kit were used to avoid contaminations. The agarose gel ensured to solidified completely before the comb is removed to prevent leaking when DNA is injected into well. The agarose gel was placed in the electrophoresis tank with the well lay nearby the negative terminal to ensure that the DNA fragments are moving in the right direction. The UV imager ensured to operate normally so that the UV light will be switched off automatically whenever the imager is opened so that it causing no harm. .

Results First Batch

1 kbp marker

100 bp marker

Figure 1

DISCUSSION This discussion will look at the overall procedure and the result obtained. For Experiment A: Lysis Buffer Hl is added to the human blood sample to mechanically break up the plasma membrane and nuclear membrane of blood cell. Proteinase K added to remove the protein framework of the chromosomes inside the nucleus which was previously enveloped by nuclear membrane. Pre-wash and Wash Buffers contain 100% ethanol to isolate scaffold DNA proteins from the mixture of chemicals by precipitation with ethanol and thus cleaned. The sample was centrifuge at maximum speed to eliminate the added ethanol completely

From experiment A, the end result obtained is purified DNA sample.

While, for Experiment B: Denaturation of DNA at 94C for 3 minutes aimed to separate the double stranded DNA by breaking the hydrogen bond between the complementary bases via heating. PCR master mixes provide the key ingredients necessary for performing PCR in a pre mixed and optimized format that streamlines and simplifies the PCR workflow. Master mixes come pre-mixed and pre-measured containing DNA polymerase, salts, magnesium, dNTPs and optimized reaction buffer. The template and primers are the only additions necessary to perform PCR. Both forward and reverse primers (forward primers- GAC TGG TTC CAA TTG ACA AGC and reverse primers- GCA AAT GGC ATT CTG ACA) added at 64C for 1 minute to anneal to the separated strands. Primers, artificial small sequences of DNA join the beginning of separated DNA strands, initiating DNA replication. DNA polymerase (enzyme responsible for DNA Replication) builds up complementary strands of DNA at 72C for another one minute. 72C is the optimum temperature for the DNA polymerase to act in the polymerase chain reaction thermal cycler. During one week storage, the DNA samples are stored in a low temperature to prevent any spoilage to the DNA samples.

For Experiment C: Restriction Endonuclease enzyme was used to chop up introns in the DNA strands into fragments (at specific base sequence known as recognition sites) that containing sticky ends. This enzyme (Hind III) cut either side of mini- or micro-satellite units leaves repeated sequences intact, giving a mixture of DNA fragments made up largely of mini- and microsatellite sequences. In a mini-satellite, a 20 to 50 base sequences will be repeated from 50 to several times. A micro-satellite sequence has 2-4 bases repeated between five to fifteen times. Same mini- or micro-satellite appear in the same part of homologous chromosomes. However, number of repeats of each satellite varies as different patterns maybe inherited. In this experiment, Hind III cuts at AAGCTT nucleotide sequence. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence : 5'GGCC3' 3'CCGG5'

This is continued with procedure D after two weeks due to some circumstances. In Experiment D: Process starts with the preparation of gel electrophoresis. Overall, the DNA fragments are placed in wells produced by the combs, in an agarose gel medium in a buffer solution to maintain a constant pH. The gel is also added with Ethidium bromide dye (EtBr) which binds to DNA fragments in the gel. Two markers (1kb and 100bp DNA ladders) were also loaded at the first and last well which used for comparison as their mass and length are known accurately. This is followed by loading of digested DNA, and DNA from PCR from each student (6 in a group). The dye can be scanned by the computer fed sensor. Another dye is also added together with the DNA samples not to bind with the DNA but to move through the gel slightly faster than the DNA so that the current can be turned off before all the samples run off the end. Electric current is passed through the apparatus and DNA fragments move at different rates (depending on their mass and charge) towards positive anode because of negative charge on phosphate group in DNA. Fragments move. Once, electrophoresis is complete, the plate is placed in a sensor that is fed into a computer.

From this whole experiment, it is proven that DNA can be extracted, amplified using polymerase chain reaction, digested by restriction endonuclease enzyme and DNA fingerprinting can be done from a blood sample. The DNA sample that have undergone polymerase chain reaction has many copies of DNA and show up in the agarose gel as a single band. The fragmented DNA should show up the bands while the control which only contain distilled water, should not have any band. However, the bands in the printout are not clear even for the DNA digested by the restriction endonuclease enzyme added (in step C). This might be because blood sample is not enough and there is only a little DNA in the blood sample since blood only contains erythrocytes as one of the components. But, red blood cells do not have nucleus to house more haemoglobin to too carry maximum oxygen to the body cells which need it, thus no DNA in red blood cells. Only white blood cells can be used to extract DNA. Besides, steps that went wrong in the procedure might have affected the result. A lot of transferring of DNA samples from reaction to tubes to tubes has reduced DNA sample a lot. Concentration of solutions used to extract must be accurate but, here, the concentrations might have altered due to long storage. The DNA also might have started to disintegrate due to long storage after the polymerase chain reaction. The control has the band because DNA sample has mixed with it during transfer into the wells of agarose gel. Thus due to some limitations, the result (figure 1) obtained is unexpected.

Limitations There are several limitations that have been identified throughout this experiment. The amount of DNA available. The analysis of image of DNA bands after undergoing electrophoresis showed that the bands were unclear and lesser than expected. This may be due to the amount of DNA available in human blood is extremely small and insignificant and furthermore the number of blood cells with nucleus containing DNA is quite little. Perhaps a larger DNA sample would be needed in order to obtain clear bands of DNA in the image. During transfer of the digested DNA, DNA from PCR and control solution (distilled water) into the well due to inappropriate and unprofessional technique. This may cause the digested DNA, DNA from PCR and control solution (distilled water) to flow out from the respective wells and cannot be detected. Centrifugation was only conducted at 12.000 rpm for one or two minutes in this experiment while a standard procedure of extracting DNA involves centrifugation at approximately 100000 rpm and the time taken is supposed to be longer, more than 20 hours in order to completely extract DNA from samples. Consequently, the chances of DNA will be much lower and minimal amount of DNA was extracted

Sources of errors Several sources of error in this experiment were identified and steps were taken to minimize these errors to make the result more accurate. Inaccuracy of precision methods. Since this experiment involves plenty of procedures which are not only lengthy but they all require accurate quantity and timing, a slight difference may be significant. Besides, inaccurate use of micropipette with teats may have caused smaller volume of DNA to be taken up and loaded into the wells when preparing for electrophoresis. Lengthy procedures call for longer handling of materials, this actually allows an extended chance and time for contamination in the DNA samples. Contamination of DNA may cause less DNA in the 20l digested DNA being analysed. Thus, the Pre-Wash and Wash Buffer solution are used to remove contaminants in DNA extraction, the procedures may have caused contamination as the sample is over exposed to the air. The agar set aside to be solidified for 30 minutes may have been longer than supposed to be. This is due to some technical errors and problems which arise due to lack of knowledge and thus, prolonging the exact time for solidification of agar. As a result, the agar may be too hard in order for the DNA fragments to move across. Thus, an appropriate time control must be apply throughout the process..

Conclusion From this experiment it is true that DNA from human blood cells can be extracted, amplified using PCR, digested into fragments using restriction enzymes and separated by gel electrophoresis. Thus, the hypothesis is accepted.

Further Investigation Another experiment can be carried out using same methods of DNA extraction, amplification, digestion and electrophoresis but apply it for other investigations by substituting human blood sample with human cheek cells, chicken liver cell and plant cells.

References 1. http://www.torahphilosophy.com/. Accessed on 14th September 2012. 2. Pearson International edition Biology, 7th edition, Campbell, Reece 2005 3. http://kmbiology.weebly.com/dna-fingerprinting---notes.html. Accessed on 14th September 2012. 4. http://wec4life.wikispaces.com/Southern+Blotting. Accessed on 14th September 2012. 5. http://learnsomescience.com/wp-content/uploads/2011/05/PCR-cycle.gif. Accessed on 14th September 2012.