Field of the Indention
I he present invention relates to the molecular iirchitecture of the human jienome in cancer and Its intersention by polymeiase chain reaction method to diireieiitKiie the genomic archiiectuiv between a health) individual and a patient with cancel
Background of the Imcntion:
Need I'or the Inxenlion
Over one billion population of India the estimated cases of cancer would be appro\imaiel> one million per year l.conomically advancing India is also constantiv changing its demogiaphic scenario and constanlly raising the bar towaids susceplibilitv cancer associated with work related stress, food habits and lack of leisure time In addiiion increasing life e\pectanc> at birth also increases proportionalely the rise in percentage of geriatric population Higher incidence ol' non-communicable diseases. especiallyh cancer is positi\el\ associated with percentage ol aged population ofa country Cancer is the world's seconil killer jfier cardiovascular disease li is alsn estimated that 9 million people will be killed bv cancer bv the end ol 2010 worldwide Ihe World Cancer Kepoil documents that cancer rates are .set to increase at an alarming rate globallv Cancer rates could increase bv 50% new cases lor the veai 2020 Details published bv Indian Council of Medical Research (ICMK) indicates that the age adjusted incidence of gall bladder cancer in women in New Delhi is 10 6 per 100 000 population and is the world's highest rate for women It is possible lo stop this loss of life bv 40"-(i through implementation of cancer prevention program and earl} diagnosis With these numbers on the forefront it is lequired that tools that can detect cancer are becoming more and more demanding I he geo-economic and geographical scenario in India demand early eancei detection tools thai can be made avalahle and affordable lo the low income group which also encj)mpasses a large poiion of the population that arc susceptible to cancer It is therelore necessarv to develop a lobust tool foi cancer detection that can be used in lemote areas with mininuim utilities and expenses Wih this philo.sophv we have created a cancer detection kit based on moleculai approaches to fulllll the needs ol demanding cancer patients in India

Description of the Related Art
4
Cancer is a class of diseases characleri/ed h> out-oi-coiitiol cell ^lowlh Iheie are o\er 100 dilTerent types of cancer, and each is elassil'ied b\ the l\pe of cell that is initialK afiecied Cancer harms the bod\' \\hen damaged cells di\ide uncoiitrollabl\ lo Ibrm lumps or masses ol tissue called tumors (except in the case ol leukemia where cancer prohibits normal blood function by abnormal ceil dnision in the blood stream) I umors can grow and inteiCere with the digeslixe. ner\ous. and circulator\ s\ stems, and llie> can release hormones that altei bod\ lunclion I umors thai stay in one spot and dcnonslrate limited growth are generall) considered lo be benign, l-'ormation ofmalignanl tumors is due to the two Ibllowing causes
A) cancerous cell manages to moNC throughout the bod> using the blood or l\mph s\ stems. destro\mg health) tissue in a process called iiuasion that cell manages lo di\ide and giow. making new blood \essels to feed itsell'in a piocess called angiogenesis
H) When a tumor successlulU spreads to other paits of liie bod\ and grows, mxading and destroNing other healthy tissues, ii is said to ha\e metastasized Ihis piocess iisell' is called metastasis, and the result is a serious condition thai is \er\ dirilcull to treat
What cau.ses cancer'
Cancer is ultimatelx the result of cells that uncontrollabl} grow and do not die Normal cells in the bod\ follow an orderly path of growth. diMsion. anil death Programmed cell death is called apoptosis. and when this process bieaks down, cancer begins to form I nlike regulai cells, cancer cells do not experience programmatic death and instead continue to grow and diMde I his leads to a mass ol abnormal cells that grows out ol contiol
Cell IS controlled b> genes in the chromosomes which is ihe master controller of all the processes of an\ living cell Ihe mot of cancer is mallunclion ol the 1)\A Since the completion of the human genome project much has been know .iboui the enin|ileMties ol the human genome and its behaxior in cancer I he abnormal growth ol" cells m cancel is therefore alUibuled to lo.ss or gam

in the chromosomal regions harboring the genes the genes that contiol cell growth vlulaiions. Meth\lation or amplifieaUon of genes damages the l)N/\ architectuie. hence leads to uncontrolled cell growth culminating to tumor I he tumor cells then grow as a lump ol tissue and will be defined b\ the cells of origin \n\ tissue with epithelial cell.s is the source of tumor generation, where the genetic changes take place and the cancer cells starts growing
DNA and Cancel Our bodies are made up ol millions ol cells Within almost e\er\ cell is 46 rod-like structures called chromosomes Chiomosomes are the packages of genetic mrormaiion that make each person unique I here are 2"^ pairs of chromosomes One chromosome ol' each pair comes from each ol'our parents Along the chromosomes aie thousands of genes, which aie made up ofDNA I here arc appio\imatel\ "^O ()()() difleivnt genes and each has an impoiiant role in the body. Oenes also play an important role in inherited disease conditions for example c\stie fibrosis, cancer and sickle cell anemia is caused b\ defects in our genes Such gene defects are called mutations, and these mutations can be passed down from a parent to a chilil Over the pa.st ten \ears. the location and the lole of hundreds of genes have been determined Ihrough researe'h. we now know that there are some genes that, when mutated, can give a persi^n an increased risk for cancer Ihese genes aie called cancer susceptibilit\ genes or "cancer genes" 0\er 100 dilTerenl cancer genes ha\e been described, and some of these have been shown to cause an increased risk foi diileieni t)pes ol eanci.i ineludmg breast cancer. o\.:i!iin cancer, colon cancer. th\roid cancer and uteime cancer
Cells can experience uncontrolled growth il there are damages or mutations to DNA. and theiefoie. damage to the genes in\ol\ed in cell dnision I oiii kc\ t>pes nl gene are responsible for the cell division process oncoeenes tell cells when to di\ide. tumor supprewor senes tell cells when not to dixidc. suicide senes control apoplosis and tell the cell to kill itself if something goes wrong, and Di\A-repair senes instruct a cell to repair damaged DNA. Cancel occurs when the cells fail to correct the miitaiions and the damage is linked to failure in apopiosis
us Patent Application 12/2.101.''I pro\ides a method lor sL-ieciing a marker gene useful lor cancer classiilcation. a method for classil>mi> cancel using the gene, a method for detecting cancer, a kit usable for the classillcalion melhud or deteclion method, and a l)\\ arra> carrMng the gene. According to the present invention, there can be obtained a gene, wherein expression ol the abo\e gene is altered independentl> from genes each ol' which expression is alteied -speciricall) during cell proliferation and expression le\el of the abo\e gene is specificallN altered depending on e\er\ ijpe of cancer samples lo be tested. \\lieieb> the classillcalion oi deteclion of cancer can be carried out eonvenienil\ and quickl\ without giMng surgical treatmenl Ihercfore. the present invention is useful for the diagnosis, ihe lieatment. and the like of cancer
I'S patent application no 12' 091.8.15 concerns nboiit a meiliods and composition winch can be used lo detect cancer in mammals, in particular in humans It nolablj desciibes serum markets oi cancers and there u.ses m diagnosis methods li also concerns tools and'or kiis which can be used to implement these methods (reagents, piobes. primers, antibodies, chips, cells etc), their preparation and their uses I he in\ention can he used i detect the presence or the progressi()n ol the cancer, particular!) breast cancer, including ai an earl> stage
DNA and Cancer Diagnosis DNA sequence inforinaiion has taken a maior leap aflei the human genome project. The .sequence mlornuiion of the DNA Irom cancer paiienls ha\e been used to define the risk of a population for susceptibilit\ towarils particular cancer, for cancel diagnosis and therap\ Sequence mi'ormalion of gene for mutation has long been used in patients to define a cancel I here are now lots of diagnostic kits axaiiable from commercial sources and being used by clinicians to confirm the pathological s\ mpioms for cancer. I he demand for such approaches has multiplied in recent >ears for more biomarkers foi various l>pes of cancer 1)\.\ based kits detecting mutations for Al'C. HKC'M. and p.S.l have been used lo detect colorectal, breast, ovarian and lung cancers I hese kits are based on genetic changes such as muialions. and poKmorphism in these specillc genes thai leads to prediciive indicator for the presence ol specific tumors in the body While all these tests are m ihe market more and more biomarkers are being developed to broaden the choice for cancel diagnostic using molecular tools I he 1)\.\ is therefore a vital source to identifv the malignanc> and the t\pe of malignaiic> ol human tissues
I he molecular approaches that are exploited lo develop kits for the identillcalion of cancer are specific for a particular type of cancer and ha\e been \er\ successful in predicting the tumor t\pe in laboratory clinical settings Infornialion regarding the cancer detection tests can be found at www cancurc.org/tests. whcrcin most of the recent tests for detection of cancel are highlighted All the.se tests are antigen specific, cancer imaging, endoscop) or ultrasound I he drawbacks of all these tests are
a) l-.xpensi\e
b) Some of the tests are positive for cancel as well as oihei altered pathologc) I he tests are time consuming
d) Computed lomograph\ Scan or CI scans exposes patients to more than 100 times the radiation of a typical chest X-ra> \loreii\ei the contrast used iii this procedure ma> cause allergic reactions.
e) Biopsy needs special procedures and sometimes ieinporar\ hospitalization While this test is ver> specific, detailed and confirmator_\ tests for the presence cancer but is onl\ test al\er the ph>sical exam, imaging, endoscopy, and laboratoi> tests for the paiieiii is indicative
Non-in\asive and cost elTectiNe tests for cancer detection ha\e therefore become \er\ iiecessar> to combat the abo\e mentioned drawbacks pariicularl\ in a countr\ where medical tests are not onl\ expensive but also not readil> axailable
SIJ.MMAKY OF TIIK INVKM ION
One embodiment oi'ihe present in\enlion coniemplaies a meiiiod comprising
(a) Obtaining blood sample of the patent to he tested, (b) deleinunmg the mean concentration of
cell free l)\A in blood plasma.(c) I he mean eoneentraiion ol cell free l)\A in plasma from a
64
hcalthx population amounts to 14 nanograms milliliter and m patients with dilTerenl neoplasias
the mean concentration detected is 118 nanogianivmilliliieis I his signillcantlx dilfereni amount
of DNA between a healthy and disea.sed indi\ uliial has also been exploited m the invention
process
Another embodiment oi'lhe present invention contemplates a method coniprisin^ (a) Obtaining the blood sample of the patient and isolating the cell free DNA from serum, (b) performing PC'R on the isolated cell free DW to which IK'KVjIX containing primers IM. P2 and P.") and other reagents are added, wherein the said l'('l<\ll.\ comprises 1120. lO.X I'CR lUilTer. Mgcl2. D.N I P. primers and laq l.n/>mc. (c) I he product ofthe Primer PI-(.S"- (JC \ CCA C \( Cri CIA CAA ICiA-.l) at ?i42 bp downstream to the stait site can enieientl) vield a PCK producloflOObp when combined with pi imerp2 (.S'-diC AlC I IC ICdCCid I ICi(iC-3) llowe\er. if Primer IT^- (.S"- ICil CAC dl \ ( (iA I I I C ( C -V) is combined with Primer PI the PCR reaction will fail to \ield an\ pioduct due to the nick in the DNA strand ofthe |i-Actin gene Ihereforc cell free DNA from health) individual will produce onl\ on PCR product from primer PI and P2. Cell iVee DNA from an indi\idiial with cancer has a dilTerent DNA iniegrit\ because the CI'D is a result ofneciosis thai \ields hirgei DNA fiagments (I ig.o) Piimer PI and P2 in combination will yield a 100 bp product tis normal indixidual. in addition primer PI and P."^ will also \ield a 400 bp product because of the presence ol intact (1-Actin gene in CID from cancer patient 1 hus the appeaiance ofthe 4(i() bp PCR product will be hallmark foi the detection of cancer in serum
Another embodiment ofthe in\ention includes a kit lor |ierforining the method described abo« L-l.xemplarN kits of the in.slanl in\eniion consist of primers specific for performing PCR reaction. I he kits may further compn.se PCR MIX reagents 1120. I OX PCR Ikifler. VIgcl2. DN I P and i aq lin/\me in suitable for performing PCR reaction and instiuction material I his inxenlion can also be used ior the follou mg purpose
1) I o detect cancer of an\ origin in huiiian bod>
2) I o screen for presence of cancer m a population based siud3) I o obserxc the recover\ of cancel after chemotheiap> or resection ofthe tumor
4) I o monitor the i-e-occurrence ofthe lumoi post therap\ or surger_5) lo identifx the elTicacN ofthe besi chemotlierap> tieatment that is responded b> the patient
BKIKF DKSCKiri lO.N OK I UK l)KA\VIN(;S
Kig.l. 1 he lin> I umor releases angiogenic faelors like VI.(ii. ICI-. IM ei as ii maiures I hesc factors lead to the formation ol'new blood \essels and pro\ide nutrients lo the giowing lumoi This also allows tumor cells and DNA from liie tumor to circulate in the blood stream I he released DNA can be detected b\ nioleculai lesis on ihe -.erum ol cancer paiients
KiR.2. Shown in the representation is the fiaiinienled of D\.\ ul |<-Actin gene foimed as a ie»«uli of apoptosis in a healthx mdi\idual 1 he primei I'l and l'2 encompassed betv\een a lOObp re^Mon will get amplified b\ I'CR. The primer IM and 1*3 will fail lo give an\ I'C'K product because this is nicked fragment
FigJ. Shown in the representation is ihe mlacl ol D\.\ ol"|i-.'\clin gene foimed as a result ul necrosis in a diseased individual I he primer PI and P2 encompassed between a lOObp region will get amplified b\ PCR In addition the primei PI and P3 will also result in a PC'K product because the inlactness of the DNA
Fig. 4 The shorthand description lor the purificatmn of cell Iree DN.'\ from serum is described All the procedures described needs to be performed at room temperature I he purified DNA can be directU used for PCK anaKsis
Fig 5 I he PCR program thai need to be used loi ilie dinplificaiion of desiivd PCK pioduci using primers PI. P2 and P3 and CM) as template
Fig6 A txpical del elctrophoresis setup that will be used lo resol\e PCR product
Fig7. A iNpical gel documentation selup thai will be used to \isuali/e PC R product
Fig 8 CI D from NI. N2 and N3 indi\ idual was purified as deseiibed in table 2 I he DNA was PCR amplified in two dillerenl reaction wiili primer PI and P2 and PI and P3
Fig 9 CI D from C I I. C 12 and C13 colorectal cancer paiieni was pun lied as described in table 2. ihc DNA was I'CR amplified in two dilVerenl reaction with primer IM and P2 and PI and 1*3 The band boxed abo\e is ihc lumor marker positive band and the bands boxed below is Ihe normal amplified product of the gene
DKTAILKI) DKSCKil'TKW OK TliK IW KM ION
An\ in\ention or disco\ery is druen b\ needs and upmost necessilN W iih this philosopln in the background the following scientific infornuiiion was used to de\ise the kit Cancer cells initiates and promotes rormaiioii ol new blood \essels and this process is i\picall\ called Angiogenesis I umor cells cannot grow be>onil I -2 mm without developing lis own blood supplj and this allows the lumor cells to procure its entire nutrient Ibi growth 1 ig I shows ihe formation of blood \essels m and around a lumor I he formation of new blood \essels allows the cancer cells to fiow in the blood stream for metastasis I his also allows the necrotic tumor cells lo release its HNA in the blood stream
13836528
Number of reports ha\e suggested detection ol lumor l)\ \ in seiuni and ha\e proclaimei! !oi tumor diagnosis. Moreover DNA extracted Ironi plasma ol cancer palienls showed the s.inie characterislics as the tumor cell DNA luilheimore. the mean DNA lex els m benign neoplasms diilcivd significanlK to that found m aggressive tumors InlereslmgK. a correlation between fiee DNA le\els and advanced disease was found in lung cancer patients I Iiiis based on e\idenees it IS likel) thai tumoi DNA found in blood ol cancer palienls signiUcanil) dillers Irom the blood o[' a health) individual and can be exploited to develop molecul.ii markers for cancer delection Serum is long known lo have cell-free circulating DN \ m minute amount but is increased in patients with autoimmune disorders, traum.i and cancel I'ariicularK in cancer llie \er\ vasculature of the blood vessels resulling Irom anglogenesl^ keeps the tumoi iissue in clo-^e contact with blood circulation I herefoie the debris ol cell de.ith b> necrosis Irom a tumor tissue will eventually dram in the blood stream and that should conlaiii DNA Cell death is a frequent event both in normal and tumor tissues Ihe only difference between these lo events is ihal in normal tissue cell deaili the fragmented DNA leleased bv the cells in the
serum are small and uniform with appro\imaicl\ the si/c of 185-200 bp While the same l)N\ released in the serum by tumor tissues through necrosis generates a spectrum of DNA Iragmenls with large si/ed strand lengths Ihe dirierences in the l)\'\ siiand length has been used as a template in the pol\merase chain reaction (I'CR) Ihe other niaior difference that has impacted in the in\cntion is the amounts of cell I'lee l)N \ in plasma or serum I he mean concenlraiion of cell free DNA in plasma from a health) population amounts to 14 nanograms milliliter and m patients with dillerent neoplasias the mean cc^iiLenliation deleeled is 118 naiioiirams/mi 111 liters I his signillcantU dillerent amount of D\ \ beixseen a health) and diseased individual has also been exploited in the invention process
Ihe PoKmerase chain reaction (PC'R) is a powerful methods to amplify tin\ amounts ol D\ \ and has now long been used to eharacteri/e l)\ \ and aKo lor diagnostic purposes As shown in ligl the P-actin gene DNA obtained liom normal indixidual is fragmented to si/e not more thanl lOObpasaresultofapoptosis I'nmer IM-(?"-(iC \ CCA ("AC C'l I C'1AC^\^ l(iA-'?)at 342 bp downstream to the start site can efllcientl> \ield a PC'K product of lOObp when combined with primer p2 (5"-(}l('Air I IC ICd ( Ci(i I lli(iC-V| Ilime\er. il Primer P">-1^'- Id! ('AC CiCA CCiA I 1 I CCC -3") is combined with I'limei i'l the i'CK ivaction will fail to >u'ld an> product due to the nick in the DNA strand olthe (I- \ctin gene I herefore (ID from health) indiMdual will produce only on PCK product Irom primei PI and P2
CI'D from an individual with cancer has a dil'lerenl DNA integril\ because the CI D is a result n\' necrosis that yields larger DNA fragments (I ig 3) Primei PI and P2 in combination will \ieid a 100 bp product as normal indi\idual. in addition primer PI and P3 will also \ield a 400 bp product because of the presence of intact |1-\ctin gene in CM) from cancer patient Ihus the appearance of the 400 bp PCR product will be hallmark lor the detection of cancer m serum
18
Cell Free DNA (CKD) Isolation Trom Scrum: Mlood contains DNA at a \ei\ low concentration and has been a maior target for genetic anal\sis lor idemifxing genetic defects m the era atkr the sequencing of the human genome Ihe circulating DNA in the blood th.it i-^ important from cancer the angle of cancer detection is the cells free DN \ I he concentration of cell free DNA (CKD) is low in plasma or seuim of health) donors but is increased in patients with tumors. Mechanisms leading to the appeaiance ol'CID in blodd remain largel) unknown lo date However, processes like apopotosis and neciosis has been shown to contribute to the

9
generation ol' Ci'l) Apoptosis is a programmed cell death mechanism and occurs m noimal epithelial cell of the intestine, colonic epithelium and uterine lining Ihese cells aie leplaced b\ the nev\ and thus are a source of CI-1) at a minimum concentration m a health\ induidual I he tumor tissue also is a conglomerate ofxarious mechanisms thai leads to its uncontrolled giowth in a \er> rapid pace While growth of these cells are in progress man\ of those cells also die because of necrosis resulting in the release ol t'l"!) m the blood stream Since this process is uncontrolled random and in massive amount the en/\mes in\ol\ed m the degiadation ol the nucleic acids become limiting leading to an increase in si/e of Ihc fiagmented l)\'\ as well as m quantit> (here are currenti) number of methods to extract /)\'A from tissues, blood and serum However, extraction of ('I'D is dilTicull because ol" die lin\ amount present in the blood I he procedure adopted in this invention is based on use ordiaotropic agent to precipitate the proteins and taking out the CM) in the aqueous la\ei In addition gbcogen is used as carrier to enhance the precipitation of the Cl-'l) b> isopropanol Ihc complete protocol is described for belter understanding ONA from 100^1 of serum was puiilled using Sodium Iodide as a chaoiiopic agent in presence of I I) IA to chealate an\ free metal u^ns present in the serum I he presence of 0 5% Sodium \-l auro>Isarcosme helps to remove the protein bound l)\A into the aquecni^ phase. Ihe precipitation of the 1)\A is greatl> enhanced b\ ihe presence of glvcogen which act', as a carrier molecule to pull down the entire l)\A in presence of isopropanol Ihe precipitated DNA is washed with 40% Lsopropanol to wash awa\ all the salts at the same time keeping the DNA as precipitate Ihe DNA obtained is brielly diied in room temperalure and then dissolved in 10-20 (il of nuclease free water and sloied at -2()"C loi furlher u.se I he D\ \ was quantified b> .spectroscopy to calculate the vield from 100 |.il of serum In biief 1 ul of the l)\ \ was diluted in sterile water to lOOjil and absorbance was measured at 260 and 280 n\l Hased on the assumption that 1 0 OD^wi equals to ."^Oug ml. the amount of DNA in each sample was calculated
Ihe amount of ('ID purified using the above mentioned method from a set of colon tumor tissue ranged from 0.01- 0 72 (.ig/^il I he large extend of the range of the CI D pertains to the stage ol the tumor from which the DNA was e.Miacted More advaiued stage ol the colon cancer vields more DNA in the serum as compared lo the DNA oinaiiied fiom the earlv stage colon tuiiioi In theorv the amount of DNA should be far less m a heallhv donor and as shown in I able I the
average amount oi'CI D extracted from normal mdi\idiial ranged from 0 01- 0 07 ^ig/ul almost 10 fold less as compared to CMl) from colon cancer patients I his method is suceessiiil to e\iract CM) from serum and the >ield orCi-'l) co-rehites with the iheoreucal assumptions and therefore is a superior method to extract CM) from serum.(Table Removed)
Table 1. The amount of DNA purified from various serum samples of colorectal cancer patients are listed. The absorbance at 260 nM was used to calculate the amount of extracted DNA. All samples listed in red colour font is from cancer patients while the serum from normal individuals are shown in yellow background.
Ihe Cl-'l) obtained b> this method was used for PCR reactions and ilesired products weie obtained under standard PVR conditions I his clearlj indicates the puritx and the mtegritx of the DNA obtained b\ this method PCR Protocol and Interpretation of the results lor call out the samples positive lor cancer(Table Removed)

Table 2. Described in this table is the amount of various reagents needed to perform the PCR reaction, fach column represents the amount of reagents needed to be added for the number of samples from 1 through 5.
Ihe following the PCR MIX is made b\ adding the reagents as shown in the I able below 1 he mix should be kept on ice toa\oid loss of ln/\ me degradation
f PCR lubes equivalent to the number of samples are taken and labeled the lubes in a
convcnienl was as the user wishes r- Pipette 2^1 of the l-X I R\C 11 I) CI I I SIR! \l I Rl I DNA (CI I)) to each P( R tubes ^ lo this is added 8)il of the PCR MIX to each tube Ihe mixture is mixed b\ xortex
followed b\ bnef centrifugation on a lablelop Ceninfuge to settle the mixture Ihe
mi.xture is kept Mack on Ice
PCR RI-.AC riON
The I'CR program that is used to obtain the desired l*CR product using CI I) as template is
shown in I'lg. 5
Keep the PCR Reaction MIX into the I'CR Machine and Run the reaction
VISIAM/AIIONOI- mi; PCR l»R()l)l CI
F- >\'eigh I 0 go!"Agarose (Molecular Uiolog) Cirade)
^ Dilute the lOX 1AL HulTer to \\ lake 10 ml of 10 I Al- Huiler and add 100 ml Double Distilled Water
^ Dissohe the I 0 g Agarose in 100 ml of I \ I Al- MulTei in a 500 ml Class conical Mask
r Microwave for 3 mm to make the mi\tuie boil
f Cool the mixture to ambient temperature Do not allow ihe Agarose to solidilx Pour the Agarose into the del electrophoresis cassette and place a 10 well comb Allow the agarose to solidify in this setup.
f Remo\e the comb and place the gel in the (iel Running \pparatus Add surHcient 1\ I \1 bulTer into the tank allowing the gel to immerse the gel
^ lake out the PCR MIX from the PCR Machine and add 2iil ofethidium biomide in each tube
^ Cenlriiuge the tubes
^ Load 5|il of the miMure in each well of the agarose gel
^ Run the gel al 90 V till the d\e travels h.iiroflhe gel
^ I ake out the gel and see the appearance of band under I \ light 1 ake pictures of the gel lor output Rl-.SrI 1
^ Ihe interpretation of the results is discussed in the next section Ihe I umor specific PCR primer pair is PI and P3 Our initial tesis on CI 1) from colon cancer patients have shown positive results in all the samples tested Ihe represenlali\e PCR Product of three noimal patients (Nl. N2. \.>) are shown in I ig S CI D Imm iioimal indi\idual \ields product with primer pair PI and P2 with a product si/e DI" 100 bp and no product with primer PI and P3 at 400 bP was observed I his technique is thereloie able lo purifj the Iragmented DN\ liom serum as a result ol'normal apoptosis in these indi\iduals lo lurihei access that the D\ \ puiified was reall> Iragmented PCR with primer PI and I'T was done and no product was
delected at a si/e of 400 bp (l-ig.8) I nder similar conditions CI \) purified from patienis with colon rectal cancer showed two distinct I'CK products with primer pair PI&P2 and P1&P3 I he PCR product obtained from primer pair PI and l'3 indicates necrotic (l-iiclin gene released from the tumors into the seru-n I'his primer pair (PI API) can be used to deteci the presence of lumor of an\ origin in the lody based on the h\pothesis made in the section desciibing the Statement of Inxenlion I he result repivscnled in lig 9 is a representali\e of 17 colorectal samples anal>/ed b\ ihe iinenied leehnique
References Cited
1) National cancer Institute I SA htlp www cancergo\■'statistics glossar2) hlip://www indiastat com'health'U) diseases-77 cancer/17811 slats asp3) P Manniuthu Projection of cancer incidence in five eiiies and cancer morialiu in India 2008.45 4-7
4) Wl l():http ''www.who int''mediacentre.news.'releases'2003.pr27''en/
5) Angioworld htlp/A\ww angioworld convcancei him
6) Arnold A. Cossman J. Hakshi \IU. JalTe IS. Waldman W and Fvorsme\er S.i Immunoglobulin-gcne rearrungemeni as unique clonal markers in human Ixmphoul neoplasms \ I ngl .1 Med 1983. "^(W. I.S'r.l.S*)^
7) w w ^ cane ure org/lests
8) Mercier H. Ciaucher C. I eugeas () and Vlu/urier C Direct PCR from whole blood without DN.A extraction \uc Acid Res. 1990.18 \2\>
9) l.con SA. Shapiro H. SkiarolT DM. and Yaros \l.l I rcc D\ \ in ihc scrum ol rancci palicnlsand ihccriccloflhcrapN Cancer Res 1977.^^7 6466.^0
10) Anker P. Mulcahy II. Vhcn XQ. and Slroun M Detection oreiiculalinji tumor DNA in the blood (plasma/scrum) of cancer patients C'ancei Metastasis 1999. 18 6^-73
11) Oiacona MH. Ruben (JC. Ic/kowski k \. Roos I H. Porter DM and Sorenson (11) Cell-free DNA in iiuman biod plasma Icni^ili measurcmeiil m patients with pancreatic cancer andhealth\ controls Pancreas 1998. 17 89-97
12) Uovnion KA. Summerha\es IC. \hiquisl DA and Shuber AP DNA inletiiii\ a«. a potcnlial marker lor stool based deleciion of coloiecial cancer Clin Chem 2()()rv49 2112-211.'?
1.1) Sarkar S. Ro\ DC, Ilatano N. AoNagi 1. (lohp k. kixama R A no\ei ank\rin repeal-containing gene (Kank) located at 9p2-4 is a growth suppressor of renal cell carcimiina .1 HiolChem 2002:277 .Vo8.S-.lfo91
14) Roy DC. Aovagi l', Sarkar S. Nomura K. Kanda II. Iwava K. lachibana VI. Kiyama R Pathological characleri/ation of Kank in renal cell carcinoma I \p Mol Pathol 2005. 78-41-48
\5) Ilalano N. Ni.shikawa \S. Mcllgunn C. Sarkar S. ()/awa K.. Shibanaka V. \aka)iina M. Ciohiji K. Knama R.
16) ■\ comprehensi\e anaUsis ol'loss ol"heiero/\gos\i_\ caused b\ hemi/\gous deleiioiis m renal cell carcinoma using a subtraction libian Mol ( aicinog 2000. .11 161-170
17) Cioebcl G. /ill M. /ill M. Miiller IIM Circulating nucleic acids in plasma or serum (CNAPS) as prognostic and predicti\e markers in patients with solid neoplasias Dis Markers 200.S. 21 10.^-120
18) Could 1-. Dlon.s II. Cahclgucnnc A. Lccoinlc 1. I.acoiirrc)c (), Mrasnu 1). Dcaiinc I', /.ucman .1. l.aurcnt-Puig P Dctcciion ol'plasma lunior l)\A in head and neck squamous cell carcinoma by micro.satellile i>pin{> and p53 mutaiion anaKsis Cancer Kes 2000. 60:707-711
19) Ihijssen MA. Swmkel.s I)W. Ruers I I. de Kok IM Dilleienee helvveen free circulalmy plasma and serum I)\A m patients with colorectal li\er metastases Anticancer Res 2002:22:42142-5.
20) Hoddv .11. Cial S. Vlalone I'R. Hams Al . Wainscoai .IS |'rospL-cli\e siud\ ol quantitation ol'plasma 1)\A levels in the diajj^nosis ofmalignant \eisus heniizn prosiale discase. Clin Cancer Res 2005. 11 I .^94-1.199
21) »odd> Jl.. Cial S, Malone PR. .Sliaida \. Wainscoai .IS, 1 larris \l 1 he role ol ceil-lree DNA .si/e distribution in the manaiiemenl ol'prosi.iie cancel Oncol Res 2006. Ki 3."^-41
22) I.opergolo A. /alTaroni N Hiomolecular markers of outcome prediction iii proolaie cancer. Cancer 2009. 11.^ .1058-.''^()67
2.1) l-.M loires.H.R. Williams and A Amon.Cieneiics 2008. 179 7.17 746
24) (irimm 1). Uauer J. Schoenberjier .1 Hlockade ol Neoanyiogenesis. a New and Promising lechmque to Control the (jrowth of Malignant 1 umors and their Metasta.ses Curr Vase Pharmacol. 2009. 7 347-v>7
25) late MC. \ghi MK l}iolog\ of aiigiogeiiesis and iinasion in glioma Neurolherapeutics. 2009. 6 447-457
26) Hormones, (iencs. and Cancel I diied b> Mrian I Henderson. Mrucc Ponder, and Ronald K Ross 450 pp . illustrated New York. Oxford I ni\cisit\ Press. 2003
While the invention has been described in detail and with leferenee to speeilie examples thereof, it will be apparent to one skilled in the art thai \arious changes and modil'icalion can be made therein without departing from the spirit and .scope thereof

Wc claim
1. A method for diagnosing cancer in humans comprising the steps of:
a. obtaining blood sample oithe patent to be tested:
b. obtaining the cell free DNA from serum:
c. determining the mean concentration of cell Iree DNA in blood plasma.
d. comparing the said mean concentration with that ol"cancer free patients:
wherein a high mean concentration indicates the probabilit\ ol'cancer.
2. 1 he method as claimed in claim 1. wherein the said mean concentralion of a health) person is 14 nanograms/milliliter.
3. Ihc method as claimed in claim 1. wherein the said mean concentralii)n of a person suflerinu from cancer is ol'the rantze of 118 luinoLirams'milliliter
4. A method for detecting cancer in humans comprising the step of'
a. obtaining the blood sample ot'ihe patient and isolating the cell free DNA from seruin.
b. performing I'CR on the isolated cell free DNA to which I'CRMIX containing primers
PI, P2 and P3 and other reagents are added:
c. anal\/ing the product corresponding to primers where product from primers PI and
P3 in addition to the product from primer P1 and P2 indicates high probabiIil\ of
cancer.
wherein the said PC'RMIX comprises 1120. l0X PCR liuffer Mgcl2. DN 1 P. primers and taq Enzyme.
5. A method as claimed in claim 4 . wherein the product obtained b> primer PI and P2 is of
100bp.
6. A method as claimed in claim 4 . wherein the producl obtained by primer P1 and P3 is of 400 bp.
7. A method as claimed in claim 4, wherein the primers are specillcall\ designed lor li-aclin gene present in human genome.
8. A method as claimed in claim 4, wherein the primer p1 is ha\ ing sequence (5"-GCA CCA CAC C T C TA CAA 1 CA-3) on |i-aclin gene.
9. A method as claimed in claim 4. wherein the primer P2 is ha\ ing sequence (5-GIC A IC TIC rCG CCIC. TTG GC-.V) on ß actin gene

10. A method as claimed in claim 4. wherein ihe primer P3 is ha\ing sequence (5-G 1 C \C GCA CGA I" IT CCC-3) on fJ-aciin gene.
11. A method as claimed in claim 4. wherein the said melliod can be performed using an\ gene of the human genome.
12. A kit for practicing the methods ordaiin 2. comprising of,
a. reagents suitable for performing poly niera.se chain reaction:
b. primer specific for performing PCK reaction on ß-aclin gene:
c. instruction material