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1L\D]$KPDG$]DGIRUDOZD\VEHLQJKDQG\VXSSRUWLQ WKH ODE DQG VWD\LQJ ZLWK PH WKURXJK WKLFN DQG WKLQ RI WKHUHVHDUFKZRUN 'U$JD6\HG6DPHHU'U=DKRRU$KPDG%KDWDQG'U -DQ 0RKG 5DWKHU IRU EHLQJ WKH PRUDO VXSSRUW HYHU  DOZD\VDQGEHLQJWKHIDFWRUZKLFKLQFXOFDWHGLQPHWKH VSLULWWRDFKLHYHP\JRDO , DP KLJKO\ WKDQNIXO WR 0U )D\D] $KPDG 'DU IRU EHLQJDOZD\VWKHUHWRKHOSPHLQWKHODEZRUN $OO P\ VHQLRUV DQG FROOHDJXHV IRU WKHLU WLPHO\ DGYLFH HVSHFLDOO\'U7DKLU0DOOD'U$GIDU107-fold) of RNA abundance can be measured, and (c) it provides insight into both qualitative and quantitative data. Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different. Traditional PCR is used to exponentially amplify target DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR. The starting template for a PCR reaction can be DNA or RNA. DNA is usually the appropriate template for studying the genome of the cell or tissue (as in inherited genetic diseases, somatic mutation in a tumor, or somatic rearrangement in lymphocytes) and for the detection of DNA viruses. For information on gene expression in a cell or tissue or the presence of genomic RNA in a retrovirus such as HIV, RNA is the appropriate template. RNA can be better than genomic DNA for detecting structural changes in long genes, since amplifying the spliced RNA transcript instead of the genomic sequence greatly reduces the length of DNA to be handled without losing any of the coding regions where clinically significant deletions may be expected. RT-PCR combines cDNA synthesis from RNA templates with PCR to provide a rapid, sensitive method for analyzing gene expression. The template for RT-PCR can be total RNA or poly (A+) selected RNA. RT 115

‡–Š‘†‘Ž‘‰› reactions can be primed with random primers, oligo(dT), or a gene-specific primer (GSP) using a reverse transcriptase. RT-PCR can be carried out either in two-step or one-step formats. In two-step RT-PCR, each step is performed under optimal conditions. cDNA synthesis is performed first in RT buffer and one tenth of the reaction is removed for PCR. In one-step RT-PCR, reverse transcription and PCR take place sequentially in a single tube under conditions optimized for both RT and PCR (Fig 3.1).

Figure 3.1: General schema of RT-PCR. (Courtesy: Methee Sriprapun)

3.6.1 First Strand cDNA synthesis: The first strand cDNA reaction can be performed as an individual reaction or as a series of parallel reactions with different RNA templates. Therefore, the reaction mixture can be prepared by combining reagents individually or a master mix containing all of the components except template RNA can be prepared. For cDNA synthesis we used Thermo Scientific Maxima first strand cDNA synthesis kit. This kit uses Maxima Reverse Transcriptase, an advanced enzyme derived by in vitro evolution of M-MuLV RT. This enzyme features high thermostability, robustness and increased cDNA synthesis rate compared to wild type M-MuLV RT. 5X Reaction Mix contains the remaining reaction components: reaction buffer, G173¶V oligo (dt)18 and random hexamer primers.

116

‡–Š‘†‘Ž‘‰› Procedure 1. All the components of the kit were thawed, mixed and briefly centrifuged before use. 2. cDNA synthesis was carried out on ice (Cool Box). 3. Constituents were added in RNAase free tubes in proportions as described in [Table 3.1] to make up a final volume of 20ul. 4. Constituents were gently mixed and centrifuged. Table 3.1: Constituents of cDNA synthesis kit.

5X Reaction Mix Maxima Enzyme Mix Template RNA Nuclease Free Water Total Volume

4ul 2ul 1pg-5ug to 20ul 20ul

5. Tubes were incubated at 25oC for 10 mins followed by 50oC for 15 mins in a thermocycler. 6. Reaction was terminated by heating at 85oC for 5 mins. 7. Synthesized cDNA was immediately put to RT-PCR analysis or stored at -20 oC.

3.6.2 RT-PCR for PML-5$5Į fusion transcripts Multiplex PCR reaction was carried out in a 25ul reaction volume using reverse and forward primers sets for bcr1, bcr2 & bcr3 transcripts shown in (fig 3.2a,b) [Table 3.2] (Van Dongen et al., 1999). 6-8ul of the PCR product was size-fractionated by electrophoresis in a 2% agarose gel stained with ethidium bromide and visualized under a UV transilluminator (Flourchem, HD2-Cell Biosciences) at 365 nm. Procedure The amplification method as described by (Van Dongen et al., 1999) was followed for qualitative RT-PCR studies for different PML-5$5Į fusion transcripts. In case of 30/5$5Į due to alternative splicing of the breakpoint cluster regions (bcr) on PML locus of chromosome 15, PCR products of various sizes ranging from 345 to 381 bp were found with 117

‡–Š‘†‘Ž‘‰› RARA-B primer which is found to be a ³universal reverse primer´ for all the three forms of translocations seen in APL (short isoform: intron 3 (bcr3); long variant isoform: exon 6 (bcr-2); and long isoform: intron 6 (bcr-1). A single product of either 381 or 345 bp was observed with PML-A1 primer that corresponded to the translocation breakpoints intron 6-bcr 1 or exon 6-bcr 2 and 376 bp products was observed with PML-A2 primer corresponding to bcr-3 whose breakpoint is located in intron 3. Primers PML-A2 & self-designed PML-RP primer set displayed an additional PCR product of 450 bp which served as an internal control (IC) and ensured good quality of cDNA used [Table 3.2]. Accordingly, the results of PCR for 30/5$5Į were interpreted as long (l), long variant (v) isoform or short (s) isoform (Fig3.2b).

(A)

Figure 3.2: A, Schematic diagram of the exon/intron structure of the PML and RARA genes, involved in t(15;17)(q22;q21).B, three types of PML-5$5Į transcripts, related tothe different PML breakpoint regions. (Courtesy: JJM Van Dongen et al., 1999)

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‡–Š‘†‘Ž‘‰› Table 3.2 : Primers for PML-5$5Į fusion transcrits (bcr-1, bcr-2, bcr-3) analysis

Tm (oC)

PML-A1

5'-CAGTGTACGCCTTCTCCATCA-3'

54oC

A1+RARA-B = (381)bp (345)bp

bcr-1 bcr-2

PML-A2

5'- CTG C T G G AGG CT GTGGAC-3'

54oC

A2+RARA-B = (376)bp

bcr-3

RARA-B

5'-GCTTGTAGATGCGGGGTAGA-3'

54oC

PML-RP ¶-CTGACTGTACCACAGCCATAGG-3¶

54oC

Transcript Product Transcript Tyre Size (bp)

A2+RP= (450)bp

Internal Control

JJM van Dongen et al., Leukemia (1999) 13, 1901±1928

Standardized PCR Program The following temperature profile was used for amplification different PML-5$5Į fusion transcripts. 1. Initial denaturation 95°C for 5 minutes. 2. Denaturation 94°C for 30 seconds. 3. Annealing 54°C for 30 seconds. 4. Extension 72°C for 30seconds. 5. Final Extension 72°C for 7 minutes. Temperature profile from step 2-4 was used for 35 cycles before final extension. Table 3.3 : Primers for p15 and b-actin semi-quantitative analysis by RT-PCR p15(F)

ࡾ-TGGGGG CGGCAGCGATGAG-ࡾ

Tm (oC)

Product Size (bp)

54oC 450 bp

p15(R)

ࡾ-AGGTGGGTGGGGGTGGGAAAT-ࡾ,

54oC

b-actin(F)

ࡾ- TGACGGGGTCACCCACACTGT-ࡾ

54oC

b-actin(R)

ࡾ- CTAGAAGCATTTGCGGTGGAC-ࡾ

54oC

680 bp

Teofili et al; Leukemia (2003) 17, 919±924

The semi-quantitative expression of p15 gene was carried out by comparing the intensity of the p15 band with band intensity of b-actin. This strategy allowed us to compare the relative p15 transcript levels in different

119

‡–Š‘†‘Ž‘‰› patient samples. Moreover, the b-actin DPSOL¿FDWLRQ served as control for the p15 RT-PCR reaction, to further verify the integrity and DPSOL¿FDELOLW\ of RNAs as well as the HI¿FLHQF\ of the reverse transcription step.

Standardized PCR Program The following temperature profile was used for p15 mRNA amplification. 1. Initial denaturation 2. Denaturation 3. Annealing 4. Extension 5. Final Extension

95°C for 5 minutes. 94°C for 30 seconds. 54°C for 30 seconds. 72°C for 30seconds. 72°C for 7 minutes.

Temperature profile from step 2-4 was used for 35 cycles before final extension.

3.7 Real TimeQuantitative Polymerasechain reaction (qRTPCR) The real-time polymerase chain reaction (real-time PCR) is a recent modification to PCR that is rapidly changing the nature of biomedical science research. It was first introduced in 1992 by Higuchi and coworkers and has seen a rapid increase in its use since (Higuchi et al., 1992, 1993). The principle of q-PCR assays is straightforward: following the RT of RNA into cDNA, it requires a suitable detection chemistry to report the presence of PCR products, an instrument to monitor the amplification in real- time and appropriate software for quantitative analysis. Real-time PCR allows precise quantification of specific nucleic acids in a complex mixture even if the starting amount of material is at a very low concentration. This is accomplished by monitoring the amplification of a target sequence in real-time using fluorescent technology. Hence as the number of gene copies increases during the reaction so the fluorescence increases. This is advantageous because the efficiency and rate of the reaction can be seen. In quantitative PCR (q-PCR), PCR products are labeled using a fluorescent reporter molecule, and the quantity of product determined by measuring the fluorescence intensity of the reaction. Fluorescent signal can be measured during the amplification process (real time q-PCR). Real Time q-PCR analysis is performed during the

120

‡–Š‘†‘Ž‘‰› exponential stage of an amplification reaction, where a direct relationship between amount of product, signal intensity, and quantity of initial template present exists. At this stage, the amount of product generated by the reaction is not limited by depletion of required reagents, accumulation of inhibitors, or inactivation of the polymerase. Detection chemistries can be either probe or non- probe based, also referred to as µspecific¶ and µQRQVSHFLILF¶ respectively. The most widely used nonprobe-based chemistry detects the binding of SYBR Green I to ds (doublestranded) DNA. In solution, the unbound dye exhibits little fluorescence; during the PCR assay, increasing amounts of dye bind to the nascent ds DNA. When monitored in real-time, this results in an increase in the fluorescence signal as the polymerization proceeds. The PCR product can be verified by plotting fluorescence as a function of temperature to generate a melting curve of the amplicon. An important disadvantage is that their specificity remains dependent on the specificity of the primers. Probe-based chemistries make use of amplicon-specific fluorescent probes and a fluorescent signal is only generated if the probe hybridizes with its complementary target. Therefore probe-based chemistries introduce an additional level of specificity, in effect combining RT-PCR with a validation step previously carried out separately after the PCR. 3.7.1 Probe Based Detection Probe based quantitation uses sequence specific DNA based fluorescent reporter probes. Sequence specific probes result in quantitation of the sequence of interest only and not all dsDNA. The probe contains a fluorescent reporter and a quencher to prevent fluorescence. Common fluorescent reporters include derivatives of fluorescein, rhodamine and cyanine. Quenching is the process of reducing the quantum yield of a given fluorescence process. Quenching molecules accept energy from the fluorophore and dissipate it by either proximal quenching or by Fluorescence Resonance Energy Transfer (FRET). Most reporter systems use FRET or similar interactions between the donor and quencher molecules order to create differences in fluorescence levels when the target sequences are detected. The fluorescent reporter and the quencher are located in close proximity to each other in order for the quencher to prevent fluorescence. Once the probe locates and hybridizes to the complementary

121

‡–Š‘†‘Ž‘‰› target, the reporter and quencher are separated. Separation relieves quenching and a fluorescent signal is generated. The signal is then measured to quantitate the amount of DNA.

3.7.1.1 TaqMan Probe TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researchers at Cetus Corporation, and technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied Biosystems for research applications. The TaqMan probe principle relies on the ¶-¶ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore based detection. As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stage of the PCR; however, the TaqMan probe significantly increases the specificity of the detection. TaqMan probes were named after the videogame Pac-Man (Taq polymerase + PacMan = TaqMan) as its mechanism is based on the Pac-Man principle.

Principle The TaqMan probe consists of two types of fluorophores, which are the fluorescent parts of the reporter protein (Green Fluorescent Protein (GFP) has an often used fluorophore). While the probe is attached or unattached to the template DNA and before the polymerase acts, the quencher (Q) fluorophore (usually a long wavelength coloured dye, such as red) reduces the fluorescence from the reporter (R) fluorophore (usually a short wavelength coloured dye, such as green). It does this by the use of Fluorescence (or Forster) Resonance Energy Transfer (FERT), which is the inhibition of one dye caused by another without emission of a proton. The reporter dye is found on the ¶ end of the probe and quencher at the ¶ end. Once the TaqMan probe has bound to its specific piece of the template DNA after denaturation (high temperature) and the reaction cools, the primers anneal to the DNA. Taq polymerase then adds nucleotides and removes the TaqMan probe from the template DNA. This separates the quencher from the reporter, and allows the reporter to give off itsenergy.

122

‡–Š‘†‘Ž‘‰› This is then quantified using a computer. The more time the denaturing and annealing takes place, the more opportunities there are for the TaqMan probe to bind and, in turn, the more emitted light is detected (Fig 3.3).

Figure 3.3: Working principle of TaqMan Probe. (Courtesy: Varity Summers)

3.7.2 Real Time Quantitation of PML-5$5Į fusion transcripts For quantification of PML-5$5Į hybrid fusion gene by real time q-PCR, we used Geno-Sen¶V 30/5$5Į (APL) Real Time PCR Kit.The GenoSen¶V 30/5$5Į PCR Reagents constitute a ready to use system for detection and quantification of 30/5$5Į using Polymerase chain reaction (PCR). The Specific Master mix contains reagents and enzymes for the specific amplification of either the bcr-1 isoform or bcr-3 isoform of 30/5$5Į fusion gene and for the direct detection in fluorescence channel Cycling A.FAM. Subsequently the Reference gene (abl) was detected on Cycling A.FAM in a separate reaction to determine ratio of fusion transcript Vs abl gene. External positive Standards (PML Bcr-1 S 1-5), (PML Bcr-3 S 1-5) & (abl S 1-5) were supplied with the kit, which allowed the determination of the gene load. Reference Gene i.e. abl allowed to determine the 30/5$5Į Vs abl ratio & simultaneously control possible PCR inhibition. The kit contained following contents in Vials having Color Coder tops to distinguish between different reagents (Fig3.4).

123

‡–Š‘†‘Ž‘‰›

Figure 3.4: Kit constituents of Geno-VHQ¶V PML-5$5Į quantitation kit. R = Reagents S = Quantitation Standards W = Molecular Grade Water.

Procedure The reaction was carried out on the Cooling Block. Desired numbers of PCR tubes were placed into the Cooling box and labeled accordingly. For each patient, tubes were labeled in duplicates (one for bcr-1/bcr-3 tanscript and second for abl gene). PCR tubes for standards & at least one negative

124

‡–Š‘†‘Ž‘‰› control (Water, PCR grade) were included per PCR run. To generate a standard curve, all supplied Standards (PML Bcr-1 S 1-5) (PML Bcr-3 S 15) & (abl S 1-5) were used for each PCR run. Before each use, all reagents were thawed completely and mixed by pipetting or by brief vortexing.

Protocol For each sample following pipetting scheme was used (Fig 3.5).

Figure 3.5: Protocol for quantitation of bcr-1 and bcr-3 fusion transcripts

Quantitation was carried out in Agilent Stratagene Mx-3000-P RealtimePCR platform. The thermal profile used is for integrated cDNA synthesis and amplification and following temperature profile was used. For cDNA synthesis: First hold Second hold

50°C for 15 minutes 95°C for 10 minutes

For amplification: 1. Denaturation 2. Annealing 3. Extension

95°C for 15 seconds. 55°C for 20 seconds. 72°C for 15 seconds.

Temperature profile for amplification from step 1-3 was used for 40 cycles.

125

‡–Š‘†‘Ž‘‰› 3.7.3 Quantitation of bcr-1 / bcr-3 fusion transcripts. The quantitation standards provided in the kit (PML Bcr-1 S 1-5), (PML Bcr-3 S 1-5) & (abl S 1-5) were treated in the same way as extracted samples and the same volume was used i.e. (2µl) instead of the sample (Fig 3.5). To generate a standard curve, all 5 Standards were used. Bcr-1 or Bcr3 fusion gene copy number as well as that of the abl gene was detected against the reference standards used. The standard curve generated reflected the working of the standards. Values of the unknown bcr-1 or bcr3 fusion transcripts were calculated as percentage of the bcr-1 or bcr-3 fusion gene/abl (reference gene). % age of fusion transcript detected = bcr-1/bcr-3 copy number/ul x100 abl copy number/ul

3.8 Extraction and Quantitation of genomic DNA High-molecular-weight DNA was isolated from blood samples using modified Phenol-Chloroform Proteinase-K extraction protocol and by Himedia DNA extraction kit.

3.8.1 Phenol-Chloroform method: (Blin and Stafford, 1976) Principle DNA is extracted from the mammalian cells by lysing the cell membranes using detergents like SDS. The protein content of the cells is then precipitated either by organic solvents like phenol-chloroform-isoamyl mixture or by various salts such as sodium chloride, ammonium acetate or potassium acetate. Finally the DNA is precipitated by ethanol or isopropanol and the DNA pellet is dissolved in Tris- EDTA. Protocol ¾ 1.5ml of EDTA preserved blood was taken in falcon tube (50ml) to which 15ml of Lysis solution was added and kept at -20°C for 30 min. ¾ The above mixture was centrifuged at 3000-4000 rpm at 4°C and decanted to discard the supernatant. ¾ The pellet left was suspended in l0 ml of saline EDTA (SE) solution and vortexed briefly to ensure that no cell clumps remained. 126

‡–Š‘†‘Ž‘‰› ¾ To the suspension, l ml of l0% SDS and proteinase K to a final concentration of 100 ȝJPO was added and incubated at 37°C in a water bath overnight. ¾ Next day, equal volume of TE saturated phenol was added and the mixture was gently mixed by inversion of tubes on overhead shaker for 15 minutes. ¾ The tube was then centrifuged at 3000-4000 rpm at 4°C for 15 minutes. ¾ The supernatant aqueous phase was collected in a fresh polypropylene tube without disturbing the interphase with the help of a micropipette fitted with a wide bored tip. ¾ To the supernatant from above step, equal volume of TE saturated phenol: chloroform: isoamylalcohol (25:24:1) was added and the mixture was shaken on overhead shaker for 15 minutes and steps 6 and 7 repeated. ¾ To the supernatant thus obtained in fresh tube, equal volume of chloroform: isoamylalcohol (24:1) was added and the tube was shaken, and step 6 and 7 repeated. ¾ To the supernatant from the above step, 1/10th volume of chilled 3M sodium acetate (pH 5.2) solution and 2.5 volume of chilled ethanol or equal volume of isopropanol were added and mixed by gently inverting the tube. If visible precipitate of genomic DNA appeared, it was transferred to 1.5ml microfuge tube and centrifuged at 6000 rpm for 5 minutes. The pellet thus obtained was washed with ȝO of 70% ethanol and re- centrifuged. The washing was repeated. ¾ If the precipitate of genomic DNA was not visible, the tube was then allowed to stand at either -70°C for 10 minutes or at -20°C for overnight. Next day, the tube was centrifuged at 6000 rpm at 0°C for 45 minutes. The pellet thus obtained was washed twice with 70% ethanol as above. ¾ Air/vacuum dried DNA pellet was dissolved in ȝO of DNA storage buffer and stored at 4°C or at -20°C for longer periods.

127

‡–Š‘†‘Ž‘‰› 3.8.2 Quantitation of DNA The concentration of the DNA obtained was measured spectrophotometer at 260nm Wavelength by using the formula:

in

a

DNA ȝJPO = A260 x 50 x dilution factor. The purity of DNA was checked by using A260/ A280 ratio. The quality of the DNA isolated from the blood samples was analyzed by 1% agarose gel electrophoresis.

3.9 Analysis of DNA; Agarose Gel Electrophoresis (AGE) Principle Electrophoresis is a technique for separation of molecules based on molecular weight and electrical charge. The matrix used in AGE is agarose with ethidium bromide dye added to it. Agarose is a linear polysaccharide obtained from seaweed and is made up of the basic repeat unit agarobiose, which comprises of alternating units of galactose and 3,6 anhydrogalactose. Ethidium bromide dye, which is added in the gel, gets intercalated between the bases of the DNA thereby converting the twisted helix to a linear structure. Upon electrophoresis with Tris Borate/acetate buffer serving as electrolyte, negatively charged DNA in the gel moves towards positive electrode (anode). Procedure 1. The edges of the gel running tray were sealed with tape to form a mould. The tray was set on a leveled bench and an appropriate comb for forming the sample slots in the gel was positioned 0.51.0mm above the plate to form a complete well when agarose is poured in the mould. 2. To 1g of agarose was added 50ml of 1x TAE buffer (see appendix) in a conical flask, and the level of the slurry was marked with a marker on the flask. The volume was sufficient to prepare a gel of 3-5mm thickness. 3. The slurry was heated in a boiling-water bath until the agarose dissolved. The volume of the solution was checked with the help of the mark made on the flask, and was replenished with water when 128

‡–Š‘†‘Ž‘‰› needed. The gel solution was allowed to cool down to about 50°C and ethidium bromide was added to a final concentration of ȝJPO The gel solution was mixed thoroughly by gentle swirling. 4. The warm agarose was poured into the sealed gel tray and allowed to set completely for 30-45 minutes at room temperature. When the gel was well set, the comb and the tape was carefully removed, and the gel was mounted in the electrophoresis tank. Electrophoresis buffer (1x TAE) was added to the tank tocover the gel to a depth of about1-2mm. 5. ȝO of DNA samples, ȝO of 6x gel-loading buffer (see appendix) and ȝO of deionized water were mixed in microfuge tubes and loaded into the wells along with a molecular size marker on one side of the gel. 6. Electrophoresis was carried out at 80 volts until dye had migrated a sufficient distance through the gel. The electric current was turned off and the gel was examined on a UV illuminator and photographed. 7. The high-molecular-weight DNA was used for further molecular investigation.

3.13 Methylation specific PCR (MS-PCR) Principle Methylation Sensitive PCR is the foremost technique which is used to study the methylation status of the CpG islands located in the promoter regions of the gene. The technique employs the differential amplification of the promoter region using specific primers designed against the methylated (5-MeC) and unmethylated cytosines after the treatment with the sodium bisulfite. The detection of 5-MeC using bisulfite conversion was first reported by (Frommer et al., 1992) and enabled by (Clark et al., 1994). Treating genomic DNA with sodium bisulfite converts the unmethylated F\WRVLQH¶V to uracils while as preserving the methylated (5-MeC) ones. The XUDFLO¶V then in turn become thymines in subsequent PCR amplification of the bisulfite modified DNA. When compared to the original sequence, the

129

‡–Š‘†‘Ž‘‰› presence of a thymine, instead of a cytosine, prior to a guanosine indicates the presence of unmethylated cytosine in the original DNA strand. Preservation of a cytosine is evidence that methylcytosine was present originally (Clark et al., 1994). Using bisulfite converted DNA, PCR is performed with primers designed to include at least two CpG dinucleotides in each forward and reverse primer. Two sets of primers are designed: One, for methylated sequences, that retains the CpG complementarity; a second, for unmethylated sequences, that is complementary to a TpG sequence. The presence of a band using the methylated primers is evidence for methylation in the original sequence (Cottrell and Laird, 2003). This technique is sensitive, but does not convey information about the density of methylation. The mechanism of the bisulfite conversion reaction involves the following steps: 1. Sulfonation: addition of bisulfite to the 5-6 double bond of cytosine. 2. Hydrolytic deamination: deamination of the resulting cytosine± bisulfite derivative to give a uracil±bisulfite derivative. 3. Alkali desulfonation: removal of the sulfonate group by a subsequent alkali treatment to give uracil. The extent of sulfonation formation is controlled by pH, bisulfite concentration, and temperature.

Figure 3.6: Schematic diagram of the bisulfite conversion reaction.

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‡–Š‘†‘Ž‘‰› The important conditions for a ³VXFFHVVIXO´ bisulfite conversion are 1. Clean and fully denatured DNA. 2. Freshly prepared bisulfite. 3. Low pH for the deamination reaction. 4. High pH for the desulfonation reaction. 5. Hydroquinoneto reduce bisulfite oxidation. After bisulfite treatment, the unmethylated cytosines will be converted to thymine residues, and the methylated cytosines will remain intact. The conversion of cytosine to uracil by bisulfite is remarkably selective and efficient when carried out under the ³VWDQGDUG´ protocol. The rate of chemical conversion has been estimated to be in the order of 99.5 to 99.7% of all cytosines, but is more often 95 to 98% due to varying DNA quality. Procedure for Bisulfite Conversion 1. ȝO of the genomic DNA (50ug) were taken in a micro centrifuge tube. 2. Then 2.2 ȝO freshly prepared 3M NaOH were added to it and the final volume was made to 20ul; tube was vortexed and centrifuged at 6000g for 1min. 3. The mixture was then incubated at 37°C for 15 min and then at 90°C for 2 min. 4. The mixture was then transferred to and centrifuged at 6000g for 1min. 5. Then ȝO of saturated sodium metabisulfite pH 5.0 (BDH) was added to the ice cold mixture and vortexed for 1 min. 6. To the vortexed mixture ȝO hydroquinone (10 mM) were added; the contents were then vortexed and centrifuged at 6000g for 1min. 7. The mixture was then overlayed with 200 ȝO mineral oil to prevent evaporation and limit oxidation and incubated for 4 to 16 h at 55°C in a water bath with a lid.

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‡–Š‘†‘Ž‘‰› 8. Mineral oil was then removed and the desalination was carried out using 3URPHJD¶s Wizard DNA Clean-Up resin according to PDQXIDFWXUHU¶s instructions. 9. The eluant was collected in micro-centrifuge tube after centrifuging the mini column at 6000g for 1min. 10.To the eluant ȝO of NaOH (3M) were added and mixture was incubated at 37°C for 15 min and then centrifuged at 6000g for 1min. 11.Finally ȝO Ammonium Acetate of pH 7.0 were added followed by ȝO chilled absolute ethanol, the contents were mixed thoroughly, and kept at ±20°C overnight. 12.The contents were then centrifuged at 14,000 rpm for 15 min at 4°C next day to pellet out the modified DNA. 13.DNA was then transferred to new microcentrifuge tube and resuspended in ȝO distilled water and stored immediately at ±20°C until further use.

3.13.1 MS-PCR for p15 Gene Promoter hypermethylation MS-PCR analysis of P15 gene was carried out for its promoter using primer sets [Table 3.4] (Chim et al., 2003). About 5.0 ul of the bisulphite converted DNA (normal and Leukemic) from each sample was used for the setting up of MS PCR. Universal Methylated Human DNA (Zymo Research, USA) was used as positive control for methylated alleles whereas DNA from normal healthy subjects was used as a control for unmethylated alleles. Water was used as a negative PCR control in both reactions. Procedure The primer sequences designed for the promoter region of P15 gene was reported previously by Chim et al., (2003). Primer sequences of P15 promoter for the unmethylated reaction amplified a 154-bp product; and for the methylated reaction amplified a 148-bp product. The annealing temperature for both the unmethylated and methylated reactions of p15 gene promoter is given in [Table 3.4]. PCR products were analyzed on 2% agarose gel as described above by Agarose gel electrophoresis. 132

‡–Š‘†‘Ž‘‰› Table 3.4 : Primers, Tm & bp size for p15 gene promoter Hypermethylation analysis

Tm (oC)

Product size (bp)

56oC

148 bp

56oC

154 bp

p15 M F: ¶- GCG TTC GTA TTT TGC GGT T -3¶ p15 M R: ¶- CGT ACA ATA ACC GAA CGA CCG A -3¶ p15 U F: ¶-TGT GAT GTG TTT GTA TTT TGT GGT T -3¶ p15 U F: ¶-CCA TAC AAT AAC CAA ACA ACC AA -3¶ Chim et al; British Journal of Haematology. 2003. 122:571±578.

Standardized PCR Program The following temperature profile was used for amplification of both unmethylated and methylated alleles. 1. Initial denaturation 95°C for 5 minutes. 2. 3. 4. 5.

Denaturation Annealing Extension Final Extension

95°C for 40 seconds. 56°C for 30 seconds. 72°C for 45 seconds. 72°C for 7 minutes.

Temperature profile from step 2-4 was used for 35 cycles before final extension.

3.14 Treatment plan for APL patients The APL patients were categorized into low risk, intermediate risk and high risk groups on the basis of their baseline leukocyte and platelet counts and subjected to treatment with ICAPL-2006 and Arsenic Trioxide (ATO) based protocols. 05 of the 33 patients categorized as intermediate risk group were treated with ATO based protocol. In addition all the relapsed patients were treated with Arsenic Trioxide (ATO) based protocol. The ICAPL-2006 and ATO protocols are outlined in [Tables 3.5 & 3.6].

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‡–Š‘†‘Ž‘‰› Table 3.5: ICAPL-2006 treatment protocol Remission Induction (All patients) DNR 60mg/m2/d (d 2, 4, 6, 8) (>70 yrs only days 2, 4, and 6) ATRA 45mg/m2/d (day 1 until CR) Dexamethasone 2.5mg/m2/12hx15 days ( If WBC> 5x109/L) Consolidation therapy (Risk adapted) Low Risk

Intermediate Risk

High Risk

:%&” 10 x 109/L

:%&” 10 x 109/L

WBC> 10 x 109/L

3OWV” 40 x 109/L

Plts> 40 x 109/L

3OWV” 40 x 109/L •\