TECHNICAL FIELD

The present invention relates to primers and methods for analyzing chimerism in conditions such as Bone Marrow Transplantation (BMT) using a broad spectrum of Variable Number of Tandem Repeats (VNTRs) or Short Tandem Repeats (STRs) or a combination thereof by qPCR (Quantitative Real Time Polymerase Chain Reaction). This facilitates a rapid screening of a number of informative markers that could serve as signature sequence for each individual, thereby functioning as an invaluable prediction tool for successful chimerism. The invention further relates to the development of a commercial kit providing a broad spectrum of VNTR and STR primers that can be used for chimerism analysis.

BACKGROUND AND PRIOR ART OF THE INVENTION

Currently the therapy of choice for many Human blood diseases like leukemia, severe aplastic anemia, lymphoma, multiple myeloma, immune deficiency disorders, acute lymphoblastic leukemia, Chronic myelogenous leukemia, cancers such as breast or ovarian is bone marrow transplantation.

Over the past 40 years, bone marrow transplantation and hematopoietic stem cell transplantation have been used with increasing frequency to treat numerous malignant and nonmalignant diseases. Post-World War II "Cold War" fears of nuclear warfare triggered a huge interest in the effects of radiation on the human body.

Bone marrow transplantation (Allogeneic) is a procedure in which a person receives stem cells (cells from which all blood cells develop) from a genetically similar, but not identical, donor. The transplantation success rate is estimated to be around 35-45%. The major causes of treatment failure are disease relapse, graft rejection and graft-versus-host disease (GVHD) phenomenon. Hence, transplantation of Bone marrow was conducted on patients who were subjected to a screen to determine the extent of tissue compatibility. This screen i.e., Human Leukocyte Antigen (HLA) typing is now considered to be a standard practice in Pre BMT in patients and donors. Although BMT is conducted on HLA matched individuals, Patients demonstrate GVHD at a percentage of 60 % in case of HLA matched individuals and 80 % in case of HLA mismatched individuals. Thus, techniques like chimerism analysis using DNA polymorphism would help to reveal the extent of donor cells in the recipient blood after the transplant thereby giving the clinicians an idea of the Graft rejection.

Chimerism analysis involves identifying the genetic profiles of the recipient & donor and then evaluating the extent of mixture in the recipient's blood, bone marrow, or other tissue. This testing (engraftment analysis) by DNA is accomplished by the analysis of genomic polymorphisms called STR or VNTR loci.

Chimerism analysis has become an important tool for pre-transplant surveillance of engraftment and also has become a routine method for documentation of engraftment after allogenic haematopoeitic stem cell transplantation (HSCT). It offers possibility to realize impending graft rejection and can serve as an indicator for recurrence of underlying malignant or nonmalignant disease. More recently, chimerism analysis has become a basis for treatment intervention, such as to avoid graft rejection, to maintain engraftment or to treat imminent relapse by preemptive immunotherapy. Haematopoeitic chimerism can be assessed based on differences of polymorphic genetic markers between the donor and the recipient.

Analysis of chimerism by polymerase chain reaction mediated amplification of STR or VNTR has become a routine procedure for the evaluation of engraftment after allogeneic stem cell transplantation. A Variable Number Tandem Repeat (VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.

A short tandem repeat (STR) in DNA occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 50 base pairs (bp) (for example (CATG) n in a genomic region) and is typically in the non-coding intron region. A short tandem repeat polymorphism (STRP) occurs when homologous STR loci differ in the number of repeats between individuals. By identifying repeats of a specific sequence at specific locations in the genome, it is possible to create a genetic profile of an individual (U.S. Pat. No. 4963663, US Pat. No 5411859, U.S. Pat. No. 6214545).

Chimerism was studied retrospectively by a PCR using limited sets of VNTR or STR and analyzing the products on a PAGE gel and the limitations that may arise are:

A. The information on the number of STR and VNTR loci is limited that could cover many informative alleles for both donor and recipient in the unrelated donor setting thereby limiting the diagnosis level of detecting an informative marker which will differentiate the recipient and donor B. Time consuming process (3-4 days) since the methodology involves PAGE (Poly - acrylamide gel electrophoresis) which is not specific and there are laborious steps involved in handling PAGE.

C. No commercial kit available for detecting chimersim analysis covering most of the informative alleles.

STATEMENT OF THE INVENTION

Accordingly, the present invention relates to a combination of primer pairs for amplifying Short Tandem Repeats (STRs) or Variable Number Tandem Repeats (VNTRs) or a combination thereof, wherein each said primer pair within the combination comprises a forward primer and a reverse primer having sequence selected from a group comprising Seq IdNos. 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,43,45,47,49, 51, 53, 55, 57, 61, 69 and 71 as forward primers; and Seq Id Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 58, 62, 70 and 72 as reverse primers; A method of identifying STRs or VNTRs or a combination thereof as informative marker for chimerism analysis, said method comprising acts of a) isolating genomic DNA from a first and a second individual and subjecting the DNA to qPCR amplification with conventional primers for amplification of predetermined STRs or VNTRs or a combination thereof and b) screening for primer pair distinguishing the amplification pattern in the first and the second individual and identifying STRs or VNTRs or a combination thereof corresponding to said distinguishing primer as informative marker for chimerism analysis; a method of analyzing chimerism using STRs or VNTRs or a combination thereof, said method comprising acts of a) isolating genomic DNA from a recipient of genetic material from a first individual, b) subjecting the genomic DNA to qPCR amplification with primer corresponding to informative STRs or VNTR or a combination thereof from the first individual as identified by method as above and c) screening by pre-BMT analysis followed by post BMT analysis with the informative marker and optionally quantifying the samples using real time PCR and interpreting the amplification result for the analysis of chimerism; a method of identifying STRs or VNTRs or a combination thereof as informative marker for chimerism analysis, said method comprising acts of a) isolating genomic DNA from a first and a second individual and subjecting the DNA to qPCR amplification with primers as above for amplification of predetermined STRs or VNTRs or a combination thereof and b) screening for primer pair distinguishing the amplification pattern in the first and the second individual and identifying STRs or VNTRs or a combination thereof corresponding to said distinguishing primer as informative marker for chimerism analysis; a method of analyzing chimerism using STRs or VNTRs or a combination thereof, said method comprising acts of a) isolating genomic DNA from a recipient of genetic material from a first individual, b) subjecting the genomic DNA to qPCR amplification with primer corresponding to informative STRs or VNTR or a combination thereof from the first individual as identified by method as above and c) screening and optionally quantifying the amplification result for the analysis of chimerism; a combination of primers as above for analysis of chimerism by qPCR; and a kit for chimerism analysis, said kit comprising a combination of primers as above and amplification reagents, optionally along with instruction manual.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

In order that the invention may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present invention wherein:

Fig 1A shows amplicons visualized in agarose gel, for samples land 4 after carrying out PCR
using markers VWA, FES and THO .

Fig IB shows visualization of amplicons of Fig 1A on a 10% PAGE.

Fig 1C shows analysis of fold change for sample 4 after subjecting to qRT PCR and comparison with sample 1 as standard.

Fig ID shows results obtained on qRT PCR of mixing sample 4 with different ratios of sample 1 to prove the concept of cycle threshold shift of the recipient pattern towards donor pattern after BMT.

Fig 2A shows amplicons of pre BMT recipient buccal swab, post BMT recipient and donor blood DNA (of case study 1) electrophoresed on 1% agarose gel after conducting end point PCR using markers listed in table 1.

Pre BMT recipient buccal swab(plsR), post BMT recipient(PlhR) and donor (PlhD) blood DNA is extracted and 75ng of DNA is used for end point PCR using 32 markers listed in Table 1. The amplicons are electrophoresed on a 1% Agarose gel and documented using UV gel documentation system.

Fig 2B shows results of Complete Chimerism by qRT PCR analysis of the donor and recipient cells (of case study 1) using M32 informative marker.

Pre BMT recipient buccal swab (PlsR), post BMT recipient (PlhR) and donor (PlhD) blood DNA is extracted. Real time PCR is carried out with 50ng of DNA using the informative marker identified in the end point PCR. Percentage chimerism is calculated by normalizing with the endogeneous control (18srRNA).

Fig 3A shows amplicons of Pre BMT recipient (from EDTA & heparin) and donor (from EDTA & heparin) blood DNA (of case study 2) electrophoresed on 1% agarose gel after conducting end point PCR using markers listed in table 1.

Pre BMT Recipient DNA is extracted from EDTA anti coagulated blood (P2eR), heparinized blood (P2hR). Donor DNA is extracted from EDTA (P2eD), heparinized blood (P2hD). 75ng of recipient and donor DNA is used for end point PCR using 24 markers listed in Table 1. The amplicons are electrophoresed on a 1% Agarose gel and documented using UV gel documentation system.

Fig 3B shows amplicons of post BMT recipient blood DNA & pre BMT donor blood DNA (of case study 2) on comparison with the informative markers identified in pre MBT analysis, electophoresed on 1% agarose gel.

Post BMT recipient blood DNA (P2hR) & donor blood DNA (P2hD) is extracted and 75ng of DNA sample is used for end point PCR using the informative markers identified in the pre BMT analysis. The amplicons are electrophoresed on a 1% Agarose gel and documented using UV gel documentation system.

Fig 3C shows results of Incomplete Chimerism by qRT PCR analysis of the donor and post BMT recipient cells (of case study 2) using M, 11, informative marker.

Post BMT recipient blood DNA (P2hRpst) & donor blood DNA (P2hD) is extracted and 50 ng is used for real time PCR using the informative markers identified in the end point PCR. Percentage chimerism is calculated by normalizing with the endogeneous control (GAPDH).

Fig 4A shows amplicons of pre BMT recipient buccal swab, post BMT recipient and donor blood DNA (of case study 3) electrophoresed on 1% agarose gel after conducting end point PCR using markers listed in table 1.

Recipient buccal swab (P3sR), post BMT Recipient (P3hR) and donor blood DNA (P3hD) is extracted and 75ng of DNA is used for end point PCR using 33 markers listed in Table 1. The amplicons were electrophoresed on a 1% Agarose gel and documented using UV gel documentation system Fig 4B shows results of Mixed Chimerism by qRT PCR analysis of the donor, post BMT recipient & recipient buccal swab cells using M4 and M5 informative markers.
Recipient buccal swab (P3sR), post BMT recipient (P3Rpst) and donor blood DNA (P3D) is extracted and 50ng of DNA is used for real time PCR using the informative markers identified in the end point PCR. Percentage chimerism is calculated by normalizing with the endogenous control (GAPDH).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a combination of primer pairs for amplifying Short Tandem Repeats (STRs) or Variable Number Tandem Repeats (VNTRs) or a combination thereof, wherein each said primer pair within the combination comprises a forward primer and a reverse primer having sequence selected from a group comprising Seq Id Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 57, 61, 69 and 71 as forward primers; and Seq Id Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 58, 62, 70 and 72 as reverse primers.

In an embodiment of the present invention, the amplification is carried out by quantitative real time PCR (qPCR).

The present invention further relates to a method of identifying STRs or VNTRs or a combination thereof as informative marker for chimerism analysis, said method comprising acts of:

a. isolating genomic DNA from a first and a second individual and subjecting the DNA to qPCR amplification with conventional primers for amplification of predetermined STRs or VNTRs or a combination thereof; and

b. screening for primer pair distinguishing the amplification pattern in the first and the second individual and identifying STRs or VNTRs or a combination thereof corresponding to said distinguishing primer as informative marker for chimerism analysis.

The present invention further relates to a method of analyzing chimerism using STRs or VNTRs or a combination thereof, said method comprising acts of:

a. isolating genomic DNA from a recipient of genetic material from a first individual;

b. subjecting the genomic DNA to qPCR amplification with primer corresponding to informative STRs or VNTR or a combination thereof from the first individual as identified by method as above; and

c. screening by pre-BMT analysis followed by post BMT analysis with the informative marker and optionally quantifying the samples using real time PCR and interpreting the amplification result for the analysis of chimerism.

The present invention further relates to a method of identifying STRs or VNTRs or a combination thereof as informative marker for chimerism analysis, said method comprising acts of:

a. isolating genomic DNA from a first and a second individual and subjecting the DNA to qPCR amplification with primers as above for amplification of predetermined STRs or VNTRs or a combination thereof; and

b. screening for primer pair distinguishing the amplification pattern in the first and the second individual and identifying STRs or VNTRs or a combination thereof corresponding to said distinguishing primer as informative marker for chimerism analysis.

The present invention further relates to a method of analyzing chimerism using STRs or VNTRs or a combination thereof, said method comprising acts of:

a. isolating genomic DNA from a recipient of genetic material from a first individual;

b. subjecting the genomic DNA to qPCR amplification with primer corresponding to informative STRs or VNTR or a combination thereof from the first individual as identified by method as above; and

c. screening and optionally quantifying the amplification result for the analysis of chimerism.

In' an embodiment of the present invention, the chimerism analysis is carried out for a condition selected from a group comprising hematopoietic diseases, leukemia, severe aplastic anemia, lymphomas, multiple myeloma, immune deficiency disorders, acute lymphoblastic leukemia, and chronic myelogenous leukemia or any combination thereof.

In another embodiment of the present invention, the recipient has received the genetic material from the first individual by transplantation.

The present invention further relates to a combination of primers as above for analysis of chimerism by qPCR.

The present invention further relates to a kit for chimerism analysis, said kit comprising a combination of primers as above and amplification reagents, optionally along with instruction manual.

The present invention relates to a method that utilizes the broad spectrum of VNTR(s) and STR(s) (Table: 1) to amplify the genomic DNA of an individual such that it could facilitate the probability of getting an informative marker that distinguish the recipient and donor at an increased extent. These VNTR and STR data are collected on the basis of the increased percentage accuracy and to cover the larger population.

In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms which are used in the following written description.

Genomic DNA: In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA.

A Variable Number Tandem Repeat (or VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.

A short tandem repeat (STR) in DNA occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 50 base pairs (bp) (for example (CATG)n in a genomic region) and is typically in the non-coding intron region. A short tandem repeat polymorphism (STRP) occurs when homologous STR loci differ in the number of repeats between individuals. By identifying repeats of a specific sequence at specific locations in the genome, it is possible to create a genetic profile of an individual.

qPCR in molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.

The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time. This is a new approach compared to standard PCR, where the product of the reaction is detected at its end.

An amplicon is a piece of DNA formed as the product of natural or artificial amplification events. For example, it can be formed via polymerase chain reactions (PCR) or ligase chain reactions (LCR), as well as by natural gene duplication.

An SYBR mix is a convenient premix of all the components, except primers, template and water necessary to perform real-time PCR using SYBR® Green I Dye. Direct detection of polymerase chain reaction (PCR) product is monitored by measuring the increase in fluorescence caused by the binding of SYBR Green dye to double-stranded (ds) DNA.

In one aspect, the present invention relates to a method that utilizes the broad spectrum of VNTR and STR (Table: 1) to amplify the genomic DNA of an individual such that it could facilitate the probability of getting an informative marker that distinguishes the recipient and

donor at an increased extent. These VNTR and STR data are collected on the basis of the increased percentage accuracy and to cover the larger population.

In another embodiment, the invention provides analysis of the informative marker in one amplification set carrying the range of VNTR and STR. The methodology uses qPCR with SYBR mix which gives the following applications:

• Definite diagnosis

• Time reduction (4 hours).

In yet another aspect, the invention provides a method for optimized detection of an informative marker for Post BMT analysis using genetic polymorphism. The applications would be:

Evaluate donor/recipient cells in patients with inadequate marrow function.

• Evaluate whether graft rejection has occurred in recipients that are candidates for a second transplant.

• Routine post-transplant documentation of the donor/recipient origin of white blood cells in peripheral blood and bone marrow.

• Define whether recurrent or new malignancy has originated from recipient or donor cells.
Assess prognostic risks of rejection and recurrent malignancy.

In another aspect, this invention can be used to screen the chimerism in most of the hematopoietic diseases like leukemia, severe aplastic anemia, lymphomas, multiple myeloma, immune deficiency disorders, acute lymphoblastic leukemia, and chronic myelogenous leukemia.
In another aspect, a commercial kit can be developed wherein the hospitals can use the kit containing the broader spectrum lyophilized primers with a standard protocol. The analysis is done using qPCR for time reduction since the other methodology takes longer time and fails to give out a conclusive data.

The Genomic DNA is isolated from the clinical sample of Recipient and donor before and after Bone marrow transplantation using a commercially available kit ( Buccal swab - Sigma GenElute™ Mammalian Genomic DNA Miniprep Kit cat no: G1N70, Whole blood – Qiagen DNeasy Blood & Tissue Kit cat no: 69504). The DNA is quantified using the UV spectrophotometer. 50 ng of the Genomic DNA is used along with 100 nM of VNTR and STR mentioned in the Table: 1 (custom synthesized), lx SYBR mix (From Fermentas Maxima™ SYBR green q PCR Master Mix (2X), ROX Solution provided 200 reactions) (i.e: lOul of 2xSYBR green is used for a 20ul reaction), autoclaved water to make up the reaction to lOul, with GAPDH as an endogenous control to ensure the amount of DNA added is same in all the wells.

The program is 95°C/10 minutes (Holding temperature), 95°C/15 seconds and 60°C/1 minute for 40 cycles and this is the point where the data is collected. This is followed by a melt curve analysis for 95° C /15 seconds, 60° C / 1 minute, 95° C / 30 seconds (point of data collection) & 60° C I 15 seconds. (This is the current program and applicable to suitable changes depending on the primer types).

The data collected is normalized with the GAPDH. The Primer (VNTR or STR) pair which distinguishes the Recipient and the donor is concluded as the informative marker. The Post Bone marrow transplantation clinical sample of the Recipient alone is taken and the Genomic DNA is isolated using the mentioned kit. 50 ng of the DNA is taken for qPCR run using the above mentioned protocol only with the informative marker and analyze the amplification for the donor pattern. This will reveal whether the graft has been completely accepted by the recipient (Complete chimerism), not accepted by the Recipient (Incomplete chimerism), Portion of the graft accepted (Mixed chimerism).

In another aspect, this invention uses qPCR methodology hence it is quantifiable in case of mixed chimerism. This will facilitate the clinicians to make a decision for the patient's condition at an early stage following bone marrow transplantation.

Standard Genomic DNA isolation and qPCR techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984); and Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-Interscience (1990).

The VNTR sequences are available in most of the research papers: Developing an algorithm of' informative markers for evaluation of chimerism after allogeneic bone marrow transplantation, Department of Haematology, Christian Medical College, Vellore, India, Chimerism Analysis of a Patient with Acute Lymphoblastic Leukemia after Allogeneic Haematopoeitic Stem Cell Transplantation: A Preliminary Report, Unit of Hematology, Cancer Research Centre, Institute for Medical Research, Kuala Lumpur, 2Molecular Diagnostic and Protein Unit, Specialised Diagnostic Centre, Institute for Medical Research, Kuala Lumpur 3Institute of Pediatrics, Hospital Kuala Lumpur, Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: the Vienna experience, Children's Cancer Research Institute, Vienna, Austria, Evaluation of STR informativity for chimerism testing - comparative analysis of 27 STR systems in 203 matched related donor recipient pairs, Medizinische Klinik und Poliklinik I, Universita'tsklinikum Carl Gustav Carus der Technischen Universita"t, Fetscherstrasse 74, Dresden, Germany.

The present invention will be appreciated by clinicians skilled in the art of Bone marrow transplantation. This will drastically reduce the risk associated with GVHD in patients undergoing BMT. Since it is rapid and cover a major number of VNTR and STR for the population it will be highly helpful for the service industries and hospitals involving in clinical diagnosis for Bone marrow transplantation. This development will facilitate the design and delivery of a commercially available kit for performing the analysis which will serve the high resolution purpose and thereby increasing the number of clinical samples screened at a short duration.

This invention uses VNTR and STR sequences from different populations. The percentages of definite diagnostic information that can be obtained using specific primers are indicated in Table 1 below.

Table 1. List of primers (STR/VNTR) used for chimerism analysis by conventional and real time PCR analysis.

In one embodiment of the present invention, the various aspects of the present invention are further elaborated with the help of following examples. However, these examples should not be eonstrued to limit the scope of the invention.

EXAMPLES

The following examples illustrate various models used to arrive at the results of the present invention. However, these are preliminary models and must not be construed to limit the scope of the present invention.

Methodology:

A] Blood DNA extraction: Qiagen DNeasy Blood & Tissue Kit cat no: 69504 1) 20 ul proteinase K is pipetted into a 1.5 ml or 2 ml microcentrifuge tube. Thereafter, 50-100 ul anticoagulated blood is added to the tube. The volume is adjusted to 220 ul with PBS, and step 2 is carried out.

2) 200 ul Buffer AL (without added ethanol) is added to the tube and mixed thoroughly by vortexing, and incubated at 56°C for 10 min.

3) To the sample, 200(0.1 ethanol (96-100%) is added and mixed thoroughly by vortexing.

4) The mixture from step 3 is pipetted into a DNeasy Mini spin column placed in a 2 ml collection tube. This is further centrifuged at 6000 x g (8000 rpm) for 1 min. The flow through and collection tube is discarded and the spin column is used from step 5.

5) The DNeasy Mini spin column is thereafter placed in a new 2 ml collection tube, and 500 ul Buffer AW1 is added, and centrifuged for 1 min at 6000 x g (8000 rpm). The flow-through and collection tube is discarded.

. 6) The DNeasy Mini spin column is thereafter placed in a new 2 ml collection tube (provided), 500 ul Buffer AW2 is added to the tube, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. The flow-through and collection tube is discarded. 7) The DNeasy Mini spin column is thereafter placed in a clean 1.5 ml or 2 ml microcentrifuge tube, and 200 ul Buffer AE is directly pipette onto the DNeasy membrane. This column is incubated at room temperature for 1 min, and then centrifuged for 1 min at 6000 x g (8000 rpm) for elution.

B] Buccal swab DNA extraction: Sigma GenElute™ Mammalian Genomic DNA Miniprep
Kitcatno:GlN70

1) The swab is cut in a 1.5ml tube containing 280ul of Resuspension solution from the kit.

2) 20 mL of the Proteinase K solution is added to the sample, followed by 200 mL of Lysis Solution C (B8803). This solution is vortexed thoroughly (about 15 seconds), and incubated at 56°C for 10 minutes.

3) 500 mL of the Column Preparation Solution is added to each pre-assembled GenElute Miniprep Binding Column (with a red o-ring), and centrifuged at 12,000 x g for 1 minute. The flow through liquid is discarded.

4) 200 mL of ethanol (95-100%) is added to the lysate; and mixed thoroughly by vortexing for 5-10 seconds.

5) The entire contents of the tube are transferred into the treated binding column from step 4. A wide bore pipette tip is used to reduce shearing of the DNA when transferring contents into the binding column. This is centrifuged at 6500 * g for 1 minute. The collection tube containing the flow through liquid is discarded and the binding column is placed in a new 2 mL collection tube.

6) Prior to first use, the Wash Solution Concentrate is diluted with ethanol as described under Preparation Instructions. 500 mL of Wash Solution is added to the binding column and centrifuged for 1 minute at 6,500 x g. The collection tube containing the flow-through liquid is discarded and the binding column is placed in a new 2 mL collection tube.

7) Another 500 mL of Wash Solution is added to the binding column; and centrifuged for 3 minutes at maximum speed (12,000-16,000 x g) to dry the binding column. The binding column must be free of ethanol before eluting the DNA. The column is centrifuged for one additional minute at maximum speed if residual ethanol is seen. Finally, the collection tube containing the flowthrough liquid is discarded and the binding column is placed in a new 2 mL collection tube.

8) 200 mL of the Elution Solution is directly pipetted into the center of the binding column; centrifuged for 1 minute at 6,500 x g to elute the DNA.

EXAMPLE 1A

Blood samples are taken from four individuals belonging to four different populations and labeled as Sample 1, 2, 3 & 4. Genomic DNA is extracted using a commercial kit and visualized in a 0.8% Agarose gel. To optimize the DNA, primer concentrations and the PCR cycling program a regular PCR is carried out with sample 1 & sample 4. The DNA is quantified and 300 ng of DNA from sample 1 & sample 4 is taken for PCR using few of the VNTR and STR as mentioned in the present invention. The markers used are VWA, FES, THO & 33. PCR is carried out in a 10 ul reaction with 2X Taq PCR mix from sigma, 7.5 picomoles/ ul of forward and reverse primers. The amplicons are visualized on a 2% Agarose gel (Fig. 1A) and FEC Tho and 33 marker on a 10% polyacrylamide gel electrophoresis (Fig. IB).

Agarose Gel electrophoresis: In the instant invention, 0.8 g agarose is weighed and added into lxTAE buffer, boiled to dissolve the agarose and poured onto the agarose gel casting tray with the comb. After the temperature comes down to room temperature Ethidium bromide of final concentration 1 ug/ml is added. The comb is removed after agarose solidified and is used to separate the PCR product. Note: The loading dye composition of the instant invention is as follows: lx TAE, bromophenol Blue, Xylene cyanol and Sucrose.

Inference:

Figure 1A: The amplification showed similar pattern in both the sample 1 & sample 4 for FES & THO marker. VWA marker showed no amplification. 33 marker showed an informative band pattern that could distinguish both the samples. To have a higher resolution of the same pattern the samples are loaded on to the 10% polyacrylamide gel. Figure IB: For both the samples VWA marker showed no amplification and FES marker showed similar pattern. THO & 33 marker showed a difference in the amplification pattern. This gives informativeness to distinguish the two samples.

EXAMPLE IB

To have more specific, definite and reliable data a qRTPCR is carried out with the same markers for the above two samples. The qRT PCR anaysis is carried out using 50ng of the Genomic DNA from sample 1 & sample 4 using ABI Prism 7500 real time PCR machine. The same markers are used at a concentration of 10 nM with 2X Maxima sybr green master mix from Fermentas and the reaction volume is made upto 10 ul. An endogenous control is included to verify the amounts of DNA sample taken. The cycling program is 95 C / 10 min (Holding temperature), 45 cycles of 95 C/ 15 seconds & 60 C/ 1 minute and the data is collected at the annealing point. The program is continued with the melt curve analysis (default program as indicated in our invention). The analysis is done by keeping sample 1 as reference (Fig. 1C).

Melting curve analysis is an assessment of the dissociation-characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity. If there is more than one product being formed during PCR the melt curve displays two peaks and if there is one amplicon it is reflected in the form of single peak.

Inference:

Figure 1C: Keeping sample 1 as reference the sample 4 showed a reduced fold change in FES, THO & 33 marker. VWA showed no amplification for both the samples. Hence there is an increase in the informativeness with the markers using qRT-PCR.

Fold change is calculated by delta ct method. The ct values obtained after real time PCR analysis for the test primer is normalized with the endogenous control. The commonly used endogenosus controls include 18SrRNA, GAPDH, beta actin, beta tubulin or HPRT. Primer amplifying 18SrRNA or GAPDH is sed as endogenous control in all the examples cited in the figures of the instant invention. The ct values obtained while amplifying the informative primes either using donor or recipient is considered as 1 on a case by case basis.

To prove the concept that qRT PCR can be used to see a switching in the recipient pattern towards the donors pattern the sample 4 is mixed with two different ratios of sample 1 and qRT PCR is carried out. To prove the concept of the cycle threshold shift towards the donor pattern after the bone marrow transplantation, sample 1 is mixed with sample 4 at two different ratios and the same qRT experiment is carried out (Fig. ID).

The normalized values before and after BMT for the recipient is compared. If the values obtained by SYBR green based real time PCR amplification for recipient is closer to donor it is considered as Complete Chimerism and if it is more towards the recipient it is considered as Incomplete Chimerism. Percentage chimerism is obtained by considering either donor or recipient fold change as 100% on a case by case basis. More than 90% donor profile is considered as Complete Chimerism while less than 90% is considered as Mixed Chimerism, donor profile less than 50% is considered as Incomplete Chimerism.

Inference:

Figure ID: The sample 4 showed a gradual shift in the fold change when sample 1 is mixed at different ratios with sample 4. This pattern is observed with FES, THO & 33 marker. VWA marker showed no amplification in both the samples. Using this technique we will be able to categorize the extent of conversion from recipient to donor type.

EXAMPLE 2

Case study 1 (Patient with Relapsed ALL Acute Lymphoblastic Leukemia) Pre BMT recipient buccal swab, post BMT recipient and donor blood DNA is extracted according to the above described protocol (under methodology) and 75ng of DNA is used for end point PCR using 32 markers listed in Table 1. The amplicons are electrophoresed on a 1% Agarose gel and documented using UV gel documentation system. The results are depicted in Fig. 2A.

Pre BMT recipient buccal swab, post BMT recipient and donor blood DNA is extracted according to the above described protocol (under methodology). Real time PCR is carried out with 50ng of DNA using the informative markers identified in the end point PCR. Percentage chimerism is calculated by normalizing with the endogeneous control (18srRNA). The results are depicted in Fig 2B.

Results: One informative marker M32 is identified using endpoint PCR analysis. M32 showed only donor's profile using endpoint and Real time PCR analysis. 100% donor profile is seen and is reported as Complete Chimerism.

Interpretation: Complete Chimerism

1. In case study 1, there is only one informative marker among 32 primers tested, which warrants the usage of multiple markers to address chimerism.

2. In case study 1, donor buccal swab is used as pre BMT sample since the buccal swab continues to exhibit recipient profile after transplant. The results indicate the specificity and the sensitivity that can be obtained with the buccal swab.

3. In case study 1, informative markers are analyzed using real time PCR and a highly quantitative data is obtained. Agarose and PAGE analysis that is conventionally used are only qualitative while the preset invention provides a chimerism platform which is quantitative.

4. In case study 1, the informative marker 32 showed a complete shift from recipient to donor profile both by conventional PCR and real time PCR thus confirming the chimeric shift to be complete.

5. In case study 1, identical results are obtained with agarose gel analysis and real time PCR analysis indicating the specificity of the platform disclosed in the present invention.

Case study 2: (Patient with Beta Thalassemia) Pre BMT Recipient and donor blood DNA is extracted according to the above described protocol and 75ng of DNA is used for end point PCR using 24 markers listed in Table 1. The amplicons are electrophoresed on a 1% Agarose gel and documented using UV gel documentation system. The results are depicted in Fig. 3A.

Post BMT recipient blood DNA is extracted according to the above described protocol. 75ng of post BMT recipient and pre BMT donor DNA is used for end point PCR using the informative markers identified in the pre BMT analysis. The amplicons are electrophoresed on a 1% Agarose gel and documented using UV gel documentation system. The results are depicted in Fig 3B.

Post BMT Recipient blood DNA is extracted according to the above described protocol.50 ng of post BMT recipient and pre BMT donor DNA is used for real time PCR using the informative markers identified in the end point PCR. Percentage chimerism is calculated by normalizing with the endogeneous control (GAPDH). Fig 3C.

Results; Five informative markers are identified (M10, 11, 29, 32 & 33) using endpoint PCR during pre BMT analysis. All 5 markers showed predominantly recipient's profile in post BMT DNA analysis using endpoint PCR. Marker 11 exhibited recipient's profile using real time PCR analysis. 98% recipient & 2% donor profile is seen indicating Incomplete Chimerism.

Interpretation: Incomplete Chimerism

1. In case study 2, both heparin treated blood and EDTA treated blood is used for analysis. DNA extracted from EDTA treated samples did not show any amplicon in multiple markers both in the donor and in the recipient indicating EDTA in the blood sample could be interfering with polymerase chain reaction. No such inhibition is seen when DNA extracted from heparin treated blood is used for chimerism analysis with all 24 markers.

2. In case study 2, the blood cell count is very less, but in the present invention, a procedure to extract out DNA by spin enrichment method is optimized for the first time, from very low blood count and PCR analysis i found to be successful.

3. In case study 2, identification of five informative markers highlights the aspect of combining both VNTRs and STRs for chimerism analysis and warrants the necessity to screen post-BMT samples using 24 markers.

4. In case study 2, identical profiles are observed with conventional PCR and real time PCR for all 5 informative markers in pre BMT analysis.

5. In case study 2, markers 10, 11, 29, 32 and 33 are used for chimerism analysis using post BMT samples. All 5 markers showed only recipient genetic profile in the post BMT sample.

6. In case study 2, marker 10, 11, 29, 32 and 33 showed only recipient profile in post BMT samples both by conventional PCR and real time PCR.

7. The percentage of chimerism estimated using real time PCR analysis is nearly identical with Ml 1 primer used for analysis indicating the reproducibility of the chimerism platform disclosed in the instant invention.

Case study 3: (Patient with Aplastic Anemia) Recipient buccal swab, post BMT Recipient and donor blood DNA is extracted according to the above described protocol. 75ng of DNA is used for end point PCR using 33 markers listed in Table 1. The amplicons are electrophoresed on a 1% Agarose gel and documented using UV gel documentation system. The results are depicted in Fig 4a.

Recipient buccal swab, post BMT recipient and donor blood DNA is extracted according the above described protocol. 50ng of DNA is used for real time PCR using the informative markers identified in the end point PCR. Percentage chimerism is calculated by normalizing with the endogenous control (GAPDH). The results are depicted in Fig 4b.

Results: Two informative markers are identified (M4 & M5) using endpoint PCR analysis. Two markers show both donor & recipient's profile in analysis using endpoint and Real time PCR analysis. 84% donor & 16% recipient profile is reported as mixed chimerism.

Interpretation: Mixed Chimerism

1. In case study 3, the identification of two informative markers highlights the aspect of combining both VNTRs and STRs for chimerism analysis and warrants the necessity to screen post-BMT samples using 33 markers.

2. In case study 3, donor buccal swab is used as pre BMT sample and the results indicate the specificity and the sensitivity that is obtained with the buccal swab.

3. In case study 3, informative markers are analyzed using real time PCR and a highly quantitative data is obtained.

4. In case study 3, the informative marker 4 shows predominantly recipient profile while marker 5 displays both donor and recipient profile by end point PCR. Real time PCR analysis confirmed the percentage of donor profile to be more than 80% in the post BMT sample from the recipient, thus confirming the chimeric shift to be mixed where both donor and recipient profile is observed.

5. In case study 3, real time PCR analysis shows an identical profile using both the primers tested.

The examples stated herein are not to be construed to limit only to BMT samples. Nonetheless, it is to be noted that the same method can be used for chimerism analysis with solid organ transplant and also with other types of allogenic transplantation.

Further, the primers designed towards the VNTRs and STRs available in the literature for identification of chimerism, may be suitably modified as required by a person skilled in the art. A skilled artisan would realize that regular mutations form part of the human evolution, due to which, VNTRs and STRs regions are also prone to such mutations, variations and/or modifications. In view of the same, the instant invention also includes any primers and/or probes arising from such mutated, varied and/or modified VNTRs and STRs.

This early diagnosis helps the physician in deciding the course of treatment for the patient. The results in case study 1, 2 and 3 show that both sex matched (Case study 3 - both recipient and donor are female) and sex mis-matched post BMT samples (Case study 1 &2 -Recipient is male and donor is female) could be used for analysis to detect the chimerism profile. The study based on all three case studies is that none of the conventionally used primer pairs (Markers depicted by primer numbers- 12, 13, 14, 15 & 17 in Table 1) are informative in all three samples tested (Ref case study 1, 2 & 3) while up to 8 primers pairs are informative while using multiple combinations of VNTRs and STRs for analysis using the chimerism platform disclosed in the instant invention. Spin enrichment method is adapted to extract DNA from samples with very low blood count. Heparin blood used in chimerism platform of the instant invention is able to amplify all primers tested while EDTA treated blood which is routinely used in all diagnosis centers does not show amplification with multiple primer pairs. Agarose and real time PCR analysis in the present invention display a nearly identical profile, while amplicons that did not amplify using conventional PCR gave confirmatory results using real time PCR due to its increased specificity. In the present invention both quantitative and qualitative results are obtained by combining conventional PCR and real time PCR. A method to calculate percentage chimerism is used in real time PCR analysis and the results concurred well with the agarose gel profile and patient's response as expressed by the physicians. Patient samples from which informative markers cannot be identified by other centers are analyzed using the chimerism platform of the instant invention and more than 2 informative markers are identified and percentage chimerism is provided to the physician. (Markers depicted by primer numbers- 12, 13, 14, 15 & 17 in Table 1 are conventionally used by other centres).

The aforementioned case studies show the versatility of the chimerism platform of the instant invention in 3 different scenarios namely:

1. Complete chimerism where the genetic profile of post bone marrow transplant (BMT) sample demonstrates a complete shift from recipient to donor.

2. Mixed chimerism where the genetic profile of post BMT sample demonstrates both donor and recipient profile.

3. Incomplete chimerism where the genetic profile of post BMT sample demonstrates only recipient profile.

The following are the advantages as per the case studies. In all three case studies the informative markers do not belong to the list of primers used routinely by other chimerism analysis centers (Markers depicted by primer numbers- 12,13,14, 15 & 17 in Table 1).

Advantages:

The advantages of our current study are the following:

1. Highly quantitative compared to the existing assays.

2. A combination of VNTRs and STRs using a real time PCR based analysis that increases the sensitivity and specificity of the assay.

3. Time involved is reduced by 50%. Chimerism analysis using PAGE takes approximately 10-12 hrs from the time of DNA extraction, qRT-PCR takes 3hrs from the time of DNA extraction.

4. A high throughput analysis cannot be done with PAGE, maximum of 10-12 samples can be analyzed by PAGE.However, using qRT-PCR of the instant invention, about 30 samples can be analyzed in triplicate within 3hrs.

We Claim:

1. A combination of primer pairs for amplifying Short Tandem Repeats (STRs) or Variable Number Tandem Repeats (VNTRs) or a combination thereof, wherein each said primer pair within the combination comprises a forward primer and a reverse primer having sequence selected from a group comprising Seq Id Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 57, 61, 69 and 71 as forward primers; and Seq Id Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 58, 62, 70 and 72 as reverse primers.

2. The combination as claimed in claim 1, wherein the amplification is carried out by quantitative real time PCR (qPCR).

3. A method of identifying STRs or VNTRs or a combination thereof as informative marker for chimerism analysis, said method comprising acts of:

a. isolating genomic DNA from a first and a second individual and subjecting the DNA to qPCR amplification with conventional primers for amplification of predetermined STRs or VNTRs or a combination thereof; and

b. screening for primer pair distinguishing the amplification pattern in the first and the second individual and identifying STRs or VNTRs or a combination thereof corresponding to said distinguishing primer as informative marker for chimerism analysis.

4. A method of analyzing chimerism using STRs or VNTRs or a combination thereof, said method comprising acts of:

a. isolating genomic DNA from a recipient of genetic material from a first individual;

b. subjecting the genomic DNA to qPCR amplification with primer corresponding to informative STRs or VNTR or a combination thereof from the first individual as identified by method of claim 3; and

c. screening and optionally quantifying the amplification result for the analysis of chimerism.

5. A method of identifying STRs or VNTRs or a combination thereof as informative marker for chimerism analysis, said method comprising acts of:

a. isolating genomic DNA from a first and a second individual and subjecting the DNA to qPCR amplification with primers as claimed in claim 1 for amplification of predetermined STRs or VNTRs or a combination thereof; and

b. screening for primer pair distinguishing the amplification pattern in the first and the second individual and identifying STRs or VNTRs or a combination thereof corresponding to said distinguishing primer as informative marker for chimerism analysis.

6. A method of analyzing chimerism using STRs or VNTRs or a combination thereof, said method comprising acts of:

a. isolating genomic DNA from a recipient of genetic material from a first individual;

b. subjecting the genomic DNA to qPCR amplification with primer corresponding to informative STRs or VNTR or a combination thereof from the first individual as identified by method of claim 5; and

c. screening and optionally quantifying the amplification result for the analysis of chimerism.

7. The methods as claimed in claims 3 to 6, wherein the chimerism analysis is carried out for a condition selected from a group comprising hematopoietic diseases, leukemia,

severe aplastic anemia, lymphomas, multiple myeloma, immune deficiency disorders, acute lymphoblastic leukemia, and chronic myelogenous leukemia or any combination thereof.

8. The methods as claimed in claims 4 and 6, wherein the recipient has received the genetic material from the first individual by transplantation.

9. A combination of primers as claimed in claim 1 for analysis of chimerism by qPCR.

10. A kit for chimerism analysis, said kit comprising a combination of primers as claimed in claim 1 and amplification reagents, optionally along with instruction manual.