GENOMIC REPORT FUNDAMENTAL MEDICAL SCIENCE 1 ________________________________________

Name : Joan Vinata Winona NIM : 07120100063 Group : B2-1 (Thursday)

Faculty of Medicine Universitas Pelita Harapan 2010

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ABSTRACT
The genomic studies, coupled with computational analyses, can be used for improving our knowledge about genes and genome function. Identification of the genes and DNA sequence variants that underlies inherited susceptibility to rare and common human diseases has been a major concern of scientists for this long to understand the molecular and cellular basis of these disease. 7 8 % of human body consists of blood. It consists of different kinds of components, include DNA. These DNAs would be used later in the experiment. The goal of all this experiments were to separate the blood components (in this case was DNA). Then, to isolate the DNA from human blood, the DNA isolation experiment was done. The concentration of the DNA would be known later using spectrophotometer instrument. Using electrophoresis, the DNA would be detected and fragments of the DNA could be seen later. PCR (Polymerase Chain Reaction) experiment, had been done to exponentially amplify a small quantity of a specific nucleotide sequence, in this case CSMD gene, in the presence of template sequence of the DNA. The nucleotide order of a given DNA fragment could be determined using BLAST. All of the results of these experiments were not achieved these goals expected completely. Such as in PCR experiment, there were no fragment on the positive control, while it should be shown. It was caused by the failures occur during the experiment was worked on, either in procedures, contamination products, or the measurements.

INTRODUCTION
Blood is a specialized bodily fluid that delivers necessary substances to the body's cells such as nutrients and oxygen and transports waste products away from those same cells. The liquid part of blood called plasma, which takes up about 55% of blood volume is mostly water (92% of total volume), and it contains blood clotting factors, sugar, lipids, vitamins, minerals, hormones, enzymes, antibodies, and other proteins. In this experiment, if the microtube doesnt filled with coagulant, the blood will be separated into blood and serum. Serum refers to plasma without clotting proteins, while the remaining proteins are albumin and immunoglobulin. The other components that present in human blood are blood cellsred blood cells and white blood cellsand platelets. Red blood cells (also called erythrocytes) have the largest amount of human blood cells (4.7 to 6.1 million (male), 4.2 to 5.4 million (female) cells per microliter erythrocytes), which consist of hemoglobin which perform in oxygen transportation, have no nuclei. They are produced in our bone marrow, and circulate about 120 days before they are recycled by the liver, while in the fetus, the red blood cells are produced in the liver. They make up 40-50% of the total blood volume. White blood cellsalso called leukocytestake part most in immune system; destroy and remove old or aberrant cells and cellular debris, as well as attack infectious agents (pathogens) and foreign substances. They are produced in the same stem cells that produce red blood cells. They are 4,000-11,000 in amount per microliter of blood. Platelets, are also called thrombocytes, important for blood clotting when the wound occurs. They change fibrinogen into fibrin. This fibrin creates a mesh onto which red blood cells collect and clot, which then stops more blood from leaving the body and also helps to prevent bacteria from entering the body. Below are the functions of the blood : Transporting the products of digestion, respiratory gases, hormones, vitamin, urea, heat, oxygen Immunological functions, which producing antibodies and antitoxins, and phagocytosis of germs Blood clotting after an open wound in order to stop bleedinga part of self-repair mechanism Regulation of pH & body temperature 3

DNA ((biochemistry) a long linear polymer found in the nucleus of a cell and formed from nucleotides and shaped like a double helix; associated with the transmission of genetic information) "DNA is the king of molecules. Deoxyribonucleic acid (DNA) is the basic genetic material of most living organisms. Although a large and apparently complex molecule, the structure of DNA is in fact astonishingly simple. A single DNA molecule consists of two separate strands wound around each other to form a double-helical (spiral) structure. Each strand is made up of a combination of just four chemical components known as nucleotides- all of which have the same basic composition. Each nucleotide consists of a sugar molecule (deoxyribose) linked to a phosphate group to form the helical backbone; different nucleotides are distinguished only by the identity of the nitrogen-based unit called the nucleotide base bonded to the sugar molecule. The four bases are: Aadenine, C cytosine, Gguanine, Tthymine. Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences. DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral called a double helix. The structure of the double helix is somewhat like a ladder, with the base pairs forming the ladders rungs and the sugar and phosphate molecules forming the vertical sidepieces of the ladder. An important property of DNA is that it can replicate, or make copies of itself. Each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. This is critical when cells divide because each new cell needs to have an exact copy of the DNA present in the old cell. A gene is the basic physical and functional unit of heredity. Genes, which are made up of DNA, act as instructions to make molecules called proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases. The Human Genome Project has estimated that humans have between 20,000 and 25,000 genes. Every person has two copies of each gene, one inherited from each parent. Most 4

genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people. Alleles are forms of the same gene with small differences in their sequence of DNA bases. These small differences contribute to each persons unique physical features. PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient so that untold numbers of copies can be made of the DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA: Two "primers", short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied; An enzyme called polymerase that moves along the segment of DNA, reading its code and assembling a copy; and A pile of DNA building blocks that the polymerase needs to make that copy. As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step -- denatauration (alteration of structure), annealing (joining), and extension -- takes place at a different temperature: Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA. Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template. Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule. With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available 5

for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially. To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses -- to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of an Egyptian mummy, establish paternity or biological relationships, etc.. Accordingly, PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material. DNA electrophoresis is a method used to sort DNA molecules by length. Pieces of DNA are suspended in a tray of gel and subjected to an electric field, which causes them to migrate toward one end of the tray. The DNA separates out into bands, with the distance from the electrode corresponding to length of the strand. The technique plays a role in identifying genes for diagnosing disease and for other forms of genetic research. The DNA to be studied is broken into individual strands using restriction enzymes that cut the DNA at specific, known locations. The DNA is mixed with a dye or radioisotope that will allow its location in the gel to be identified. The individual strands are then separated from each other using DNA electrophoresis. The process begins by injecting the separated genetic material into wells that have been cut in the end of a slab of gel. An electrical field is then applied to the gel slab. DNA has a negative electric charge, and is attracted to the positive electrode. The gel resists the DNA as it moves; smaller pieces have an easier time moving through the pores in the gel and so travel further. Given a known gel preparation and electrical application, the length of a segment can be very precisely determined by the distance that it travels. It can then be cut from the gel using a scalpel. If the fragments to be separated are very short, a polyacrylamide gel is used. For longer segments, an agarose gel is used. Agarose is made from seaweed, while polyacrylamide is a synthetic polymer. The agarose gels are much less dense than the polyacrylamide gels and allow larger molecules to move through. For very long DNA segments, a recently developed method called pulsed-field gel electrophoresis must be used. That process uses an electrical field that constantly undergoes subtle 6

changes in direction to keep very long strands oriented correctly as they move through the agarose. Gel electrophoresis can be used to identify the presence of DNA strands of known length. It can thus be used to determine the existence of certain traits or genetic diseases within a given individual. DNA electrophoresis can also be used to isolate strands of DNA for recombination as part of a genetic engineering project. It can also be used to create a genetic profile of an individual for the purposes of identification, as in paternity tests or criminal forensics. DNA gel electrophoresis is a process used to separate proteins and nucleic acids in molecular biology. The gel is usually composed of a crossed-linked polymer and acrylamide which aid in separating and analyzing different parts of a DNA molecule. DNA gel electrophoresis is commonly used in forensics to determine the specific sequence of DNA to help find the leading suspect. It is also commonly used in genetics and the fields of molecular biology and biochemistry. Nucleic acids and proteins carry either a positive or negative charge. When placed in an electric chamber in DNA gel electrophoresis, each electrical charge will move toward either end of the chamber. The negative electrical charged molecules will move to the positive end of the chamber, while the positive electrical charged molecules will move to the negative end. The gel separates each of these molecules so they evenly spread throughout the reading. The gel is composed of long fibers which prevent the molecules from moving to aid in analyzing the individual molecules. Southern blotting is a technique used in DNA gel electrophoresis to determine the presence or absence of a specific nucleotide sequence in a DNA molecule. Northern blotting is a different technique used to identify gene expression in the presence of RNA, or ribonucleic acid. Eastern blotting is used to determine the presence of lipids, proteins and carbohydrates in a DNA sequence. To simply detect proteins in a DNA sequence, the technique of western blotting is used in the electrophoresis chamber through gel isolation. DNA gel electrophoresis is used quite often in forensics. It is used to separate blood proteins and DNA found at a crime scene to determine correlations with the available suspects. Gel electrophoresis is also used to determine a specific inheritance within genetics, as certain DNA and proteins are associated with different races. It is also used to solve paternity cases to determine 7

the relative relationship between individuals. DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. By generating a DNA sequence for a particular organism, you are determining the patterns that make up genetic traits and in some cases behaviors. Sequencing methods have evolved from relatively laborious gel-based procedures to modern automated protocols based on dye labelling and detection in capillary electrophoresis that permit rapid large-scale sequencing of genomes and transcriptomes. Knowledge of DNA sequences of genes and other parts of the genome of organisms has become indispensable for basic research studying biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes. The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.

MATERIALS AND METHODS Blood Separation

The materials used in this methods are blood, vacutainer, syringe, vials, and centrifuge tubes. First step, 2 samples of blood are taken from 2 different students. Each samples taken are about 8-9 ml, and put them into each vial. Second, each vial is labeled by the name of each students. Next, from each vial, 1ml of blood sample are taken, and put into the vacutainer and dont forget to label it. After that, the vacutainer are centrifuged at 3300 g, 20 oC, in 10 minutes. After it has been centrifuged, the serum must be transferred into a new vial using pipette. Last, serum must be stored at -80 oC.

DNA Isolation
The materials used in this experiments are Ethanol absolute, isopropanol (2Propanol), proteinase K, Sodium acetate, sodium dodecyl sulfate ( SDS 10% ), TrisEDTA, buffer (pH 8.0), Chloroform / Isoamyl alcohol 24 : 1, Proteinase K (which 100mg of it has been dissolved in 10ml H2O for 30 minutes at room temperature, Aliquot and store -20 oC). The first step is the vacutainer tubes contained blood sample which has been frozen at -70
o

C are taken. And then, 0.5ml of blood sample is transferred to a

microcentrifuge tube. 0.8ml 1X SSC buffer is added, and then mix it using vortex. After that, the tube is centrifuge it for 1 minute at 12000 rpm in a microcentrifuge. Then, 1ml of the supernatant must be removed and discarded into disinfectant. 1ml more of 1X SSC buffer added, and mix it again using vortex. The tubes are centrifuged for 1minute at 12000 rpm, and then all of the supernatant must be removed. Then, 375 ul of 0.2 M NaOAc is added to each pellet by using pippete, and vortex it. 25ul of 10% SDS and 5 ul of proteinase K is added into the tube, vortex, and incubate for 1 hour at 55 oC. Next, 120ul phenol/chloroform/isoamyl alcohol is added and vortex it for 30 minutes. The tube is now centrifuged for 2minutes at 12000rpm. The aqueous layer must be carefully transferred to a new 1.5 ml microcentrifuge tube, and added by 1ml of cold 100% ethanol. Mix and incubate it for 15 minutes at -20oC. The tube is centrifuged again for 2 minutes at 12000 rpm, and 9

then the supernatant must be decanted and drained on the toilet paper. Now, the tube is added by 180ul 1X TE buffer, vortex it, and incubate at 55 oC for 10minutes. After that, 20ul 2M of sodium acetate is added, and the tube must be mixed. Now, 500ul of cold 100% ethanol is added, and mix it again. The tube should be centrifuged for 1 minute at 12000rpm in a microcentrifuge, then the supernatant must be decanted and the pellet is rinsed with 1ml of 70% ethanol. For 1minute at 12000rpm, the tube must be centrifuged again in a microcentrifuge. After the tube has been centrifuged, the supernatant must be decanted and the pellet must be dried until dry. The pellet is now added with 200ul of 1X TE buffer to resuspend it, and incubate the tube at 55oC for 30 minutes. Now, to dissolve the genomic DNA, the tube must be vortex periodically. Last, the samples are stored at -20oC.

Quantitation of DNA Concentration

Materials used in this experiment are TE buffer, DNA sample, spectophotometer BIORAD, and cuvettes. The DNA sample is diluted by ratio 1:5. The cuvette is filled with 50ul of1X TE buffer. The cuvette must be set on cuvette holder of spectrophotometer, and the type of Nucleic acid is selected. Then, the spectrophotometer is blanked with 1X TE buffer. Now, the cuvette is inserted with DNA sample to read the absorbance ( 50ul final volume). The concentration is recorded. The absorbance must be fallen between 0.1 to 1.0 for accurate concentration measurement.

DNA Detection using Electrophoresis

Materials needed in this experiment are an electrophoresis chamber and power supply, gel casting tray, sample combs, electrophoresis buffer, loading buffer, ethidium bromide, and DNA marker. The gell tray is prepared. 1 gr agarose is mixed in 50ml TAE buffer in an Erlenmeyer flask. Then, the solution is boiled in the microwave. After the 60 oC temperature is reached, 1ul ethidium bromide is added. After the agarose gel is prepared, the agarose is poured off in the tray. The comb is set, and let the tray with agarose for 30minutes in room temperature to allow gel become hard. While the electrophoresis 10

chamber is being prepared, the agarose gel can be loaded. 5ul of DNA and 1ul of loading dye are added to a 0.5 ml tube, using a pipette, mix it. then, the mixture can be added to the agarose gel well according to the sample number. After that, 6ul of DNA marker is loaded, and it can be filled with TAE buffer until it reached about 1mm above gel surface. The lid must be closed, and the electrode is connected to the power supply. The electrophoresis is run at 100 V, 6 Watts, 0.6 A for about 30minutes. Now the gel can be taken out and wash it under tap water. The picture is saved using Versa DOC instrument.

Polymerase Chain Reaction (PCR)

The materials we used in this experiments are DNA template, primers, 2.5mM dNTP mix, taq DNA polymerase, 5X taq buffer, 25mM MgCl2, and dH2O. First step, a reaction mixture is set in a PCR tube by mixing 9.875ul of dH 2O, 5ul of 1X buffer PCR, 2ul of 2.5mM dNTP mix, 1.5ul of 25mM MgCl2, 0.125ul of taq DNA polymerase, 0.75ul of 10uM forward primer, 0.75ul of 10uM reverse primer, and 5ul of DNA template, for 25ul total volume. Second, the PCR tube is put in the PCR machine, by setting the program for : 1. step 1 = 95oC for 10minutes 2. step 2 = a. denaturation = 94oC for 30 seconds b. annealing = 63.5oC for 30 seconds c. extension = 72oC for 1 minute Step 2 is set for 40 cycle. 3. step 3 = a. final extension = 72oC for 10 minutes b. storage = 4oC for indefinite time Now the machine can be run, and the PCR product must be kept at 4oC.

DNA Sequencing
Material used in this experiment are computer, bioinformatic software tools for editing sequence file, and file DNA sequence in pasta format. First, computer must be turned on, and the bioinformatic software tools (cromas lite). Then, cromas lite is operated, 11

and open the file DNA sequence fasta format. If the direction has been reversed by DNA sequence, switch it to forward direction. DNA sequence is analyses, if the N base along Dna sequence has been found, it must be changed with base according to appropriate colour. After the sequence has been edited, save it. now, the sequence has to be copied in fasta format. Next, website

http://www.ncbi.nlm.nih.gov/ was opened. Nucleotide blast menu is clicked. After that, the edited DNA sequence must be paste into Enter Query Sequence Area. Search set is chosen. Human fenomic database is clicked, and then BLAST is clicked.

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RESULT 1. Blood Separation

In this experiment, after the blood sample had been centrifuged, we would get the 3 layer of blood in the microcentrifuge tube. The first layer contains plasma, the second layer contains buffy coat (white blood cell or leucocytes and platelets), and the third layer contains red blood cell or erythrocytes.

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2. Agarose electrophoresis for DNA Isolation

Figure 2. DNA isolation The left side on the figures shows the DNA marker. The odd number of the well (1,3,5,7,9,11,13) shows the first sample (Joans sample). While the even number of the well (2,4,6,8,10,12,14) shows the second sample (Karinas sample).

3. Quantitation of DNA concentration

1:5 -> DNA = 1/5 x 50ul = 10ul TE buffer = 4/5 x 50ul = 40ul DNA Concentration (mg/ml) = (OD : ) x dilution factor Purity index = A260/A280 DNA is considered pure if the purity index is 1.80 - 2.00

Joan A260 A280

I 0.80 0.26

II 0.078 0.026

Average 0.079 0.026

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Karina A260 A280

I 0.522 0.358

II 0.521 0.356

Average 0.5215 0.352

Joan (sample 1) : Purity DNA = A260/A280 = 3.03 not pure (RNA contamination) DNA concentration = (OD : ) x dilution factor = 0.079 x 5/20 = 0.01975 mg/ml

Karina (sample 2) : Purity DNA = A260/A280 = 1.46 not pure (protein contamination) DNA concentration = (OD : ) x dilution factor 0.5215 x 5/20 = 0.130375 mg/ml

From the result above, it can be concluded that both of the result are not pure, because DNA is considered pure if the purity index is 1.60 2.00.

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4. Agarose electrophoresis for PCR

Figure 4. DNA Detection Using Electrophoresis The right lane shows the DNA marker to be a control for the other samples. The third well from the left shows the positive control (should be shown), the second well shows the negative control (shouldnt be shown), the third was the first sample, the forth was the second sample. This picture shows that there are no lane at all, which means the B2-1s experiment has failed.

5. BLAST of DNA Sequence

Figure 5. BLAST

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Figure 6. Percentage of experiment succeed

Figure 7. DNA sequence

DISCUSSION
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From the first experiment, blood separation, the vial had been filled with anticoagulant, so that after the blood sample had been centrifuged, it was separated into three parts. The top or the first part was the plasma, with yellow color, the second part in the middle was the buffy coat, which contained white blood cells or leucocytes, and the third part at the bottom, which red colored, contained red blood cells or erythrocytes. The range of plasma > erythrocytes > buffy coat of total blood volume. Our result is appropriate with the theory. Normally, plasma makes up 55% of our bloods volume, buffy coat, includes leucocytes and platelets, normally about 1%, and erythrocytes normally make up 40%-50% of the total blood volume. (Blood Components. Daniel Oneil. C1999-2008 [updated Oct 23 2008]. Available from :

http://anthro.palomar.edu/blood/blood_components.html). The second experiment is DNA isolation. This experiment has a purpose to separate the DNA from protein, lipid, and other molecules. To see the DNA bands, agarose gel, which had been colored by ethidium bromide (to enable visualization of the fragments within the gel), was used together with electrophoresis instrument (Betty AF, Daniel FS, Alice SW. Diagnostic Microbiology. 12 th ed. St. Louis, Missouri: Mosby Elsevier. 2007). The DNA itself, was taken from the plasma or white blood cells, not from the whole blood. The gel would later present a number of thick and thin bands. On the figure above, the odd number on the well, which were filled with sample 1 (Joans blood sample), shows thin bands, which means the concentration of the DNA is low, because it was contaminated, either by protein or RNA. The even number on the well, which were filled with sample 2 (Karinas blood sample), which means the concentration of DNA was high, and it contained larger number of DNA than the sample 1 had. The B2-1s DNA samples were taken from Joan and Karina. From the sample one, after being measured, the results showed the concentration of Joans DNA was 0.001975 mg/ml, and the purity index showed 3.03. The second samples results for DNA concentration was 0.130375 mg/ml, and the purity index showed 1.46. Based on those measurements, both of the sample were not pure. The first samples result for purity index was above 2.00, which means it was contaminated by RNA, while the second samples result was 1.46, below 1.6, the minimum range for DNAs pureness, which means it was contaminated by protein. This impureness might be caused by mistaken when mixing the sample, or maybe 18

it was contaminated by phenol or the chloroform while taking the aqueous layer (supernatant). The second time using electrophoresis instrument, was for PCR experiment, and it was the same as the DNA isolation, using an agarose gel which had been visualized by ethidium bromide and using an electrophoresis instrument. But now, the bands shown on the figure was different from the previous. On the figure above, there was no lane at all. The positive control should occur on the picture, and the negative control shouldnt occur. So it means that our experiment was failed.

Figure 7. A1-1s result of electrophoresis for PCR.

Compared with A1-1 group, they were succeed in comparing with DNA ladder, and there wasnt any negative control occurred. The failure might be caused by the incorrect while mixing the mixture, maybe the wrong volume, or the wrong substance, the mixture was too long outside, or the enzyme used had been damaged. In DNA sequencing, experiment used was dideoxy method, or chain method, r Sanger method. The software BLAST was used to get the information about the nucleotide order of a given DNA fragment. The result shows that the similarity is almost 100%, which is 97%. 19

REFERENCES
http://anthro.palomar.edu/blood/blood_components.html http://www.purchon.com/biology/blood.htm http://wordnetweb.princeton.edu/perl/webwn?s=dna http://www.ucadia.com/uca/u12/121800.htm http://ghr.nlm.nih.gov/handbook/hgp/ http://www.medicinenet.com/pcr_polymerase_chain_reaction/article.htm http://www.wisegeek.com/what-is-dna-electrophoresis.htm http://www.dnasequencing.org/

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