BAB 1 : PENGENALAN

Pendidikan adalah sebuah aspek vital yang dapat mempengaruhi aspek-aspek lainnya. Jika tidak ada pendidikan, maka tidak akan ada kita pada saat ini dengan segala macam kecanggihan teknologi, dengan segala macam rumit masalah yang mencekik dan tidak akan ada pemimpin di dunia ini. Allah SWT menciptakan Nabi Adam as , dan hal yang pertama Allah berikan padanya untuk bekal kehidupan adalah pendidikan. Allah memberitahu beliau segala sesuatu yang belum dia ketahui sehingga akhirnya dia menjadi tahu. Allah SWT juga memberikan wahyu pertama pada Nabi Muhammad SAW berupa 5 ayat pertama dari surat Al-alaq yang mana isinya adalah perintah untuk membaca. Perintah membaca disini ertinya, agar kita sebagai manusia mengutamakan pendidikan yang bertujuan untuk memajukan hidup kita sendiri. Untuk memartabatkan institusi pendidikan, terlebih dahulu kita perlu memartabatkan golongan pendidik yang menjadi tunjang utama sistem pendidikan. Tema Hari Guru 2010,Guru Pembina Negara Bangsa adalah bersesuaian dengan peranan guru yang signifikan dalam pembangunan umat sejagat. Seperti kata mutiara Sasterawan Negara Usman Awang, Lahirnya seorang Perdana Menteri, doktor, peguam, dan pensyarah adalah bermula daripada seorang guru biasa yang mengajarnya baca dan tulis. Dengan kata lain, tanpa guru, siapakah kita? Makanya,untuk melahirkan individu yang berkualiti, guru atau pendidik mestilah terdiri daripada golongan yang benar-benar berkualiti.

PERNYATAAN MASALAH

Keperluan menjalankan kajian ini adalah di dorong oleh perasaan ingin tahu setelah mengkaji dan meneliti artikel Benarkah Guru Punca Masalah Murid/Pelajar di Sekolah, coretan Sang Tuah dalam blognya. Berdasarkan artikel ini, 60% responden menyatakan YA. Antara alasan yang di berikan respondan ialah guru malas, tidak mengambil berat tentang pelajar bermasalah, pengajaran yang membosankan, tiada persediaan, garang, selalu maki hamun dan denda pelajar. Lebihan 40% yang menyatakan TIDAK adalah mereka yang terdiri daripada guru dan guru besar.

"Kita tidak begitu suka untuk mengakui kebenaran jika ianya mendedahkan kesilapan kita.Tetapi jika kita ingin memperbetulkan sesuatu, kita perlu mengenalpasti sebab yang sebenar dan bertindak memperbetulkan kesilapan itu." ~Dr. Mahathir Mohamad Dalam pelan Induk Pembangunan Pendidikan (PIPP) Kementerian Pelajaran Malaysia 2006-2010, salah satu fokus penting ialah usaha Memperkasakan Sekolah Kebangsaan (SK). Antara ciri yang dikatakan bagi sekolah perkasa ini ialah pemimpin dan guru mestilah cekap, terlatih dan berkualiti. Apakah senarionya kini? Cuba teliti dan kaji bagaimana gaya pengurusan, bagaimana kebolehan para pengurus pendidikan ini menangangi perubahan? Bagaimana wujud tidak kepemimpinan melalui teladan dan sebagainya. Begitu juga aspek kualiti guru di sekolah; bagaimana atau apa ciri-cirinya yang kita boleh kata guru yang berkualiti atau guru cekap itu? Cekap bukan bererti mampu habiskan sukatan pelajaran tetapi tahap penguasaan atau kemahiran murid tidak meningkat? Guru-guru terlatih pula jangan sampai ada berlaku sindrom guru "letih" dan tiada arah tujuan sebagai "guru sebenar guru"? Begitu juga yang kita kata guru berkualiti itu adakah sekadar memiliki diploma/ijazah sudah berkualiti? Bagaimana tahap "ilmunya" jika tidak mampu memperbetulkan kelemahan murid dalam pelbagai aspek P&P seperti aspek ejaan, sebutan, tulisan atau lain-lain bidang kemahiran yang berkaitan? Bagaimana boleh dikatakan seseorang guru itu guru yang berkualiti jika ketrampilan ilmunya dipertikai oleh muridnya sendiri. Email seorang pelajar seperti tersiar di dalam akhbar The Star (25.03.2007) menerangkan maksudnya dengan jelas: "I am in Form One. I would like to write about my English language teacher.I think my friends and I speak better English than her. She cannot even pronounce simple words, for example, the word "crow" is pronounced as "crew". Most of the time she speaks as if she is a saleperson at the supermarket. Her command of the language is very bad. When we read correctly, she stops us and repeats the word wrongly.She confuses us most of the time. She always tells us to talk only in English and not Tamil or Malay but she speaks in Malay.I think she should go for tuition to improve her English. My friends and I often make fun of her but she doesn't understand a thing. I feel sorry for her." ~ Shureen Raj
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Bercakap tentang pengalaman saya sebagai seorang guru (2005-2010), pelbagai karenah guru dapat saya perhatikan. Dari segi personaliti, kebanyakan guru sememangnya amat menitikberatkan hal ini, mengikut etika yang telah ditetapkan. Dari segi P&P, terdapat segelintir guru yang masuk ke kelas sekadar memenuhi kehendak jadual dan silibus tanpa menghiraukan tahap penguasaan pelajar. Teknik P&P yang digunakan tidak dipelbagaikan seperti mana yang telah dipraktikkan semasa mengikuti kursus pendidikan. Akibatnya, pelajar hilang minat untuk belajar di dalam kelas ,sebaliknya lebih gemar pergi ke tempat tuisyen. Tidak dinafikan, guru kini dibebani dengan pelbagai kerja sampingan. Walaubagaimanapun, dalam menyelesaikan kerja-kerja sampingan tersebut, kualiti guru dari semua aspek perlu dimantapkan. "Kurang kreativiti dan kegagalan mengurus dengan baik oleh pihak sekolah terutama guru besar dikenalpasti punca utama pelajar Melayu di Pulau Pinang terkandas dalam pelajaran, sekaligus gagal bersaing dengan pelajar kaum lain." Prof.Dr. Aminah Ayob (Dekan Pusat Pengajian Ilmu Pendidikan, USM) ketika itu dilapor sebagai berkata bahawa berdasarkan pemerhatian pihak mereka di beberapa buah sekolah di luar bandar di negeri tersebut mendapati pentadbiran sekolah gagal mencorakkan pembelajaran murid-murid apabila guru tidak hadir di sekolah, dan mendapati (di sebuah sekolah rendah) pelajar berkeliaran dalam kelas dan bermain-main sesama sendiri\ berikutan guru kelas tidak hadir pada hari berkenaan. "Apa yang dikesalkan tiga kelas tersebut ditinggalkan begitu sahaja tanpa pengawasan. Murid-muridnya tidak buka beg ataupun membaca buku." (Utusan Malaysai 5.02.2003)

Sekolah di dirikan bukan untuk mencetak ribuan ijazah, melainkan mencetak ribuan tangantangan mahir untuk menjadi generasi penerus bangsa yang jujur, yang adil, yang istiqomah, dan yang bermartabat. Apa ertinya selembar ijazah tanpa satupun keahlian dan kejujuran yang ia -pelajar- miliki? Kebanyakan orang lupa apa tujuan mereka belajar. Mereka, lupa apa tujuan mereka di sekolahkan. Yang mereka tahu ke sekolah untuk dapat ijazah setelah itu bisa kerja. Tapi pada dasarnya tujuan sekolah bukan hanya itu.

1.2 Objectives

The aims of this study are:

To create a precise and sensitive of species specific primer of beef and pork for PCR assay. To have species specific primers that are useful for assessments of authenticity and quality of meat. To successfully isolate pork from minced meat. To detect the sensitivity and quality of pork present in minced meat. To protect customers from being fraudulent of meat products. To produce a simple, reliable and rapidly detection method of meat adulteration.

1.3 Skop Kajian Skop kajian meliputi 23 orang pelajar program Pengsiswazahan Guru Sekolah Rendah (PGSR).

1.4 Significance of Research The significant of this research is to help customers from fraud. Successful of this research will bring lots of benefits in food technology area aside to help Muslim from being cheated. In addition, it is important to make sure food are in good quality to avoid any inconvenient in future.

CHAPTER 2 : LITERATURE REVIEW

10 Food adulterations

Food adulteration can be defined as a process where quality or nature of food is disrupted through addition of foreign or inferior substances and the removal of a vital element. More worst, in some cases, the addition or mixing unnecessary substances to food could affect customers health and become harmful. In 1793,Fredick Accum, a German chemist was the first to raise the alarm about food adulteration. He had publish A treatise on adulterations of food and culinary poisons in 1820 to show danger of food adulteration to consumers. For example, in 1860,there are case where alum is used by baker to whitened brown undaunted to fraud customers. Nowadays, problems in food adulterations are still occur. This is because of the attitude of some corrupted traders. Definitely, food adulteration in meat industry brings lots of problem for meat producers and customers.
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bread. Baker did this because most

Englishmans dislike brown bread. So, by thinking about money merely, unmoral trader

(Chen,Hsieh,&Bridgman,1998;Dennis,1998;Toorap,Murch, & Ball,1997a,1997b). Traders replace the high cost of meat with the low cost meat because of the difference prices of meat. More worst, this is not a new phenomena.(Barai, Nayak, Singhal, & Kulkarni,1992;Sugarman,1985). Pork has become a choice of corrupted traders since it have similarity in colour and texture with higher value meat such as beef and lamb .[5] More than that, it is cheaper and able stable for long time storage period compare to others type of meats. or cultural conflicts as reported by Swatland (1985). Detection whether food taken are free from any authentication and adulteration is important especially for Muslim. Since Malaysia had been declare as Islamic country, so, victory of this research will able to help government in order to avoid food adulteration especially in meat industry. As state in Quran, Muslim cannot eat pork. By having pork as their meal, Muslims will be declare as doing something opposite to religion. This is proven by Al-Quran Surah 2: Verse 173 with: He has only forbidden you dead meat, and blood, and the flesh of swine and that on which any other name hath been invoked besides that of Allah. But if one is forced by necessity, without willful disobedience, nor transgressing due limits, - then is he guiltless. For Allah is oft-forgiving, most merciful.
[6]

By exchange food

composition legally, customers might exposed to serious allergic reaction or cause religious

Thus ,it is important for food producers, manufacturers and food service provider to be more sensitive and responsible when labelling their products. Otherwise, Muslims are exposed to fraud and sin. Muslim also need to be more alert to avoid them from consumed processed food claimed to be halal but adulterated with non-halal ingredients and additives. Halal and haram are important for in order to become an obedient Muslim. As state in AlQuran Surah Al-Maidah : Verse 87-88 with: O ye people! Eat of what is on earth, Halal and good; and do not follow the footsteps of the Evil One, for he is to you an avowed enemy. 2.2 Meat
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Meat can be defined as whole or part of carcass of any buffalo, camel, cattle, deer, rabbit or sheep that is slaughtered other than in the wild state or any other animal that is permitted for human consumption. However, the definition and classification of meat are not included eggs or fish. Meat is the main source of protein as well as providing energy. By having meat, several disease such as heart disease could be avoid. have meat in their diet. Generally, meat can be classified into white meat and red meat. Most mammals are classified as red meat.[8] The term red meat in culinary refers to meat colour when it is raw. It is red darker-coloured meat, contrast with white meat. White meat refers to any lightercoloured meat. The examples of red meat are sheep horses and beef. White meat are consist of poultry and pork.[9] The colour classification are based on different location and uses of the muscles. Dark meat can be found on animals legs. At this part, there are muscles design to develop endurance for long-term use and contain large amount of myoglobin. Thus, allowing muscle to use oxygen more efficiently for aerobic respiration. However, the exact definition of meat are varies by time, place, and culture. For example, meat of young mammals such as veal and milk-fed lamb are considered as white meat. In this study, beef are included in red meat category while pox are included in white meat group. Uniquely, pox will consider as red meat if cooked[10]. 2.3 Polymerase Chain Reaction (PCR) In 1953,Dr Watson and Crick had came out with theory that biological significance of the DNA molecule with its complementary base pairing that suggested a possible coping mechanisms for genetic material. One strand of DNA will be a template for the formation of a new complementary chain. By the end of copying process of DNA, two DNA ladder that identical in every way will be obtained. Then, in 1983,Dr Kary Mullis had develop a theory called Polymerase Chain Reaction (PCR). PCR work based on three simple steps known as denaturation, annealing and extension. In denaturation, double strand DNA will became single strands. Annealing steps involving primers to each original strand for syhthesis new strand of DNA. Lastly, the extension of new DNA strand from primers take place. In addition, PCR require Thermus aquaticus (Taq polymerase) to help in synthesizing new DNA strand repeatedly.[11]
[7]

Besides that, it have a

good taste when cooked in varieties of style. Thus, it is not weird when most people like to

As state above, PCR require primers in order to be well operated. Thus, selection of suitable primers are important. The more DNA sequence information are available, the better chances to find ideal primer pair . The best base pair of efficient DNA amplification is around 200-400 bp of DNA. However, it is more difficult to choose primers for efficient amplification of longer DNA fragments. More than that, the ability of primers to form stable duplex with the specific site on target DNA, no duplex formation with another primer molecule and no hybridization at any other target site is compulsory to be consider in order to gain needed results.

2.4 Gel Electrophoresis PCR products need to be analyze in order to interpret its results. Often, gel electrophoresis and agarose gel are used for the analysis. Reasons behind the choice of agarose gel electrophoresis are it is fast, easy and allow quick estimation of the purity and concentration of PCR product. The DNA fragments is separated based on molecular size. During electrophoresis, the smaller DNA fragments migrate more farther compare to more bigger fragments. Compare to arylamide, agarose gel will give better resolution and much more precise estimate of product size. In addition, this method will be help by ethidium bromide (EtBr) for checking the size and purity of a PCR product before use it in other application such as cloning or labelling. Actually, it is a dye that able bind to double stranded DNA and it can fluoresces upon excitation with UV light. In other words, EtBr are use to visualize DNA in an agarose gel. As the primers bind to fixed position within the target sequence, the expected size for PCR product is the distance between the forward and reverse primers. Size of amplicon or DNA band is extrapolated from the DNA standards included in the gel. [12] 2.5 Applications This study reveal the importance of detection of species origin of meat and meat product even in mixture meat. It is applied before that in identification of pork(Calvo,Osta,&Zaragoza,2002),beef(Piknova Kuchta,2002),ostrich(Colombo,Viacava,& Giaretti,2000)and mutton, chevon & and

chicken(Irfan-Ilhak& Arslan,2007;MMane et al.,2007)in mixed meat. Thus, previous research proven that PCR and gel electrophoresis method can be applied to detect presence of pox in beef. Detection will be interpret by looking at the band results. This is a most suitable
8

and valuable tools to identify the meat species in presence of other meat species DNA(Hopwood et al.,1999). This PCR analysis will amplify cyt b gene of mitokondrial(mt) DNA of beef(Bos taurus) and pork(Sus scrofa).Previously, the mitokondrial 12S ribosomal RNA(mt 12S rRNA) gene was used by Rodriguez et al.(2004) in PCR assay for detection of pork in raw and heat-treated meat mixture. In addition, Tanabe et al.(2007),successfully detect the amount of pork DNA in processed food. Pork meat and its fat in meat mixture also successfully identified based on D-loop mtDNA by Montiel-Sosa et al.(2000).All of these experiment proven that the most suitable part of DNA to be amplify is mitokondrial. Mitokondrial DNA (mtDNA) was targeted to design as species-specific primer

because it is maternally inherited. Normally, there are only one allele exists in an individual and no sequence ambiguities are expected from the presence of more than one allele(Unseld et al.,1995).This phenomena makes mtDNA unique as a powerful tool for tracking species ancestry, expecially in highly conserved region such as cyt b gene region (Brown,George,&Wilson,1979).Since mitokondrial gene are present in thousand copies per cell(Greenwood & Paboo,1999),the increasing of probability to achieve it as suitable DNA fragments for identification of specific-species are high (Bellagamba et al.,2001).Moreover, by using mtDNA as a marker are proven to be more efficient compare than using nuclear markers for the identification and authentication of meat (Hopwood,Fairbrother,Lockley,&Bardsley,1999).In addition, mtDNA evolve much more faster than nuclear DNA and this condition make it as the best choice(Wolf,Rentsh,& Hubner,1999).

CHAPTER 3: EXPERIMENTAL DESIGN 3.1 Procedure Below is the flow chart of the experiment that will be conducted:

3.2 Details of Materials and Methods 3.2.1 Samples Beef (bovine) and pox (porcine) samples are purchase from local wet market in Skudai, Johor. Before extraction of DNA, sample are kept at -20C to prevent enzymatic degradation. Minced of bovine and porcine are prepare for 6g in variable ratio as shown in below table (n=5): Bovine (%) Porcine(%) 3.2.2 DNA extraction Commercial kit name GenElute_Mammalian Genomic DNA Extraction Kit(Sigma) will be used for DNA extraction. About 6g of minced meat are obtained by using scalpel blade before meat is incubated for 65C in 8 ml CTAB buffer (0.055 M CTAB, 1.4 M NaCl, 0.1 M Tris and 0.02 M EDTA, pH 8) containing 50 g/ml proteinase K (Sigma).Then,after 1hour,samples are cooled and 400L of the supernatant used to extract the DNA by follow the kits instruction. 3.2.3 Polymerase Chain Reaction (PCR) 3.2.3.1 Oligonucleotide Primers 100 0 90 10 70 30 50 50 30 70 10 90 0 100

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In order to start PCR analysis, a pair of primers for both bovine and porcine are require. The first PCR primers used for porcine(Por 1) is established from GenBank database with 1.6 of Clustal W (Higgins,Bleasby,& Funchs,1992,Kesmen,2005) with 227bp).
[13] [14]

.Primer that establish by Lahiff et al., (2001),was use as this first primer for bovine (Bov1).

The second primers for both porcine (Por 2) and bovine 2(Bov2) were published by Dalmasso et al.(2004) with 290bp and 104 bp respectively. the primers sequences.
[15]

All this primers sequences

were blast in http://www.ebi.ac.uk to confirm there are no crossing over occurs between all

Table indicates details of first Oligonucleotide Primer Primer Fwd Bov 1 Rv Bov 1 Fwd Por 1 Rv Por 1 Sequence GCC ATA TAC TCT CCT TGG TGA CA GTA GGC TTG GGA ATA GTA CGA CAT TCG CCT CAC TCA CAT TAA CC AAG AGA GAG TTC TAC GGT CTG TAG Length 23 21 23 24

Primer Fwd Bov2 Rv Bov2 Fwd Por 2 Rv Por 2

Sequence GAA AGG ACA AGA GAA ATA AGG TAG GCC CTT TTC TAG GGC A CTA CAT AAG AAT ATC CAC CAC A ACA TTG TGG GAT CTT CTA GGT

Length 21 19 22 21

Table indicates details of second Oligonucleotide Primer

3.2.3.2 PCR amplication Table below represent thermal cycling process for PCR assays:
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PCR steps Initial denaturation Denaturation Annealing Extension Final extension

Temperature (C) 96 C 94 C 60C 72C 72C

Time duration (minutes) 5 1 1 1 1

After initial denaturation, cycles are repeated for other 39 times. The amplification will be run by using 25l in 0.2 ml pcr tubes containing PCR buffer

[20 mM TrisHCl (pH 8.4), 50 mM KCl], 2 g/ml BSA, 2.5 mM MgCl2, 200 M of each dNTP, 0.5 M of each primer, 2 units of Taq DNA polymerase and 100 ng DNAtemplate.

3.2.4 Gel Electrophoresis 1.5% agarose gel or 2% MS agarose (Roche) for competitive PCR in 0.5x TBE buer (0.045 M Trisborate, 0.045 M boric acid, 0.001 M EDTA, pH 8.0) and stained by ethidium bromide will be used to analyst results obtained from PCR. Results obtained from electrophoresis will be compared with 100 bp-ladder (Life Technologies).In addition, spectrophotometer can be use to check the purity of genomic DNA by taking the optical density (O.D).

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CHAPTER 4 : EXPECTED RESULTS There are list of expected results:

The precise and sensitive species specific primer of beef and pork are obtained through the PCR assays. The species specific primers obtained from the experiment are useful for meat authenticity. Pork are successfully isolated from minced meat. The detection of sensitivity and quality of pork present in minced meat are success. Cases of fraudulent towards customers are decrease. Method propose are simple, reliable, and rapid detection for meat adulteration.

CHAPTER 5 : LIST of REFERENCES Ali Arslan, O. Irfan Ilhak, Mehmet Calicioglu(2004). Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique using mitochondrial DNA.Meat Science 83 (2009) 5761
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B.G. Mane, S.K. Mendiratta, A.K. Tiwari (2009).Polymerase chain reaction assay for identification of chicken in meat and meat products. Food Chemistry 116 (2009) 806810. Bruce A. White. (1993). PCR protocols. Current Methods and Applications. New Jersey: Humana Press Inc. Chandrika Murugaiah, Zainon Mohd Noor, Maimunah Mastakim, Lesley Maurice Bilung, G.Sabudin,2009,The Usage of PCR for Meat Authentication. Degree Thesis. Universiti Teknologi Malaysia J. Scott Smith, Y.H. Hui. (2004). Food Processing. Principles and Applications. Iowa: Blackwell Publishing. Jinap Selamat, Son Radu(2008).Meat species identification and Halal authentication analysis Meat Science 72 (2006) 326330 Miguel A. Rodrguez, Teresa Garca, Isabel Gonzlez, Pablo E. Hernndez and Rosario Martn(2004). TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures. Meat Science 70 (2005)113-120 Nicolai Z. Ballin, Finn K. Vogensen, Anders H. Karlsson(2009).Species determination Can we detect and quantify meat adulteration? Meat Science 83 (2009) 165174 R. Wissiack, B. de la Calle, G. Bordin, A.R. Rodriguez (2002). Screening test to detect meat adulteration through the determination of hemoglobin by cation exchange chromatography with diode array detection. Meat Science 64 (2003) 427432. S. Ghovvati, M.R. Nassiri,S.Z. Mirhoseini, A. Heravi Moussavi, A. Javadmanesh. (2008).Fraud identification in industrial meat products by multiplex PCR assay. Food Control 20 (2009) 696699 Y.B. Che Man, A.A. Aida, C.M.V.L. Wong, A.R. Raha, R. Son. (2004). Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. Meat Science 69 (2005) 4752.

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Z. Kesmen, F. Sahin, H. Yetim.(2007). PCR assay for the identification of animal species in cooked sausages. Meat Science 77 (2007) 649653 Zhang Guoli, Zheng Mingguang, Zhou Zhijiang, Ouyang Hongsheng and Lu Qiang (1998). Establishment and application of a polymerase chain reaction for the identification of beef. Meat Science 51 (1999)233-236

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GANTT CHART Task 5 Distribution of FYUP title Collecting sample FYUP1 Briefing Writing Proposal Submission Proposal Borrow books from library and search Start writing proposal Submit first draft to the 6 7 8 Month 9 10 11 12 1 2 3 4

journals from supervisor on internet on week 3. food adulterations, PCR etc Week 4Preparing final draft proposal

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Research Proposal Presentation

Preparing slide show for presentation

Research / Experiment

Writing Report Log book, practical, Attendance

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