FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
The Patents Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION ,
(See section 10 and rule 13) '
1- TITLE OF THE INVETION: A process for the preparation of DNA from thalassaemics
blood and cost-effective and sensitive kit thereof.
2-APPLICANT (S)
(a) NAME: C.S.R.D.
(b) NATIONALITY : Indian
(c) ADDRESS : Centre for Scientific Research & Development (CSRD), People's Group, By-
pass Road, Bhanpur, Bhopal-462037, MP, India
3-PREAMBLE TO THE I In order to develop treatment and to prevent
DESCRIPTION haemoglobinopathies,it is crucial to diagnose the
haemoglobinopathies, it is crucial to diagnose the
various haemoglobinopathies. This mass screening
will help not in epidemiological survey but also in
genetic counseling. As genetic counseling is
indivisible form of genetic diagnosis. Many
pathological and molecular test have been developed
to detect haemoglobinopathies. PCR is paramount
and contemptible method to detect various
haemoglobinopathies along with mutation. DNA the
master Key of all the molecular analysis, play vital
role in PCR based study. The admirable quantity and
quality of DNA from the less amount of blood
sample is indispensable. The present study focused
on extraction of DNA from 1ml blood of
thalassaemics. This procedure is cost effective and
can be used in laboratory with limited funds in
developing countries.
PROVISIONAL COMPLETE
The following specification describes the Complete specification provided
invention.

4-DESCRIPTION Title of the Invention
A process for the preparation of DNA from thalassaemics blood and cost-effective and sensitive
kit thereof.
Field of the Invention
The Haemoglobin disorders are most common clinically serious single gene disorders in the
world. Haemoglobin disorders were significantly endemic in 60% of 229 countries, potentially affecting 75% of births, but are now sufficiently common in 71% of countries among 89% of births. Around 1.1% of couples worldwide are at risk for having children with a haemoglobin disorder and 2.7 per 1000 conceptions are affected. Every year approximately 10,000 children with thalassaemia major are born in India which constitutes 10% of the total number in the world. Among the structural variants, HbS, HbD and HbE, 25% traits are of sickle cell disease. Anaemia is very common in all the haemoglobinopathies in which the haemoglobin level is very low. To analyze the haemoglobinopathies a very significant quantity (= 10 ml) of blood sample required for extraction of DNA from anaemic patients. Molecular analysis is the golden key for detection of various haemoglobinopathies in developing countries. The extraction and isolation of DNA from blood is a necessary step for any PCR-based analysis. Hence, the method of DNA extraction is a key factor in determining the overall efficiency and reliability of any PCR-based test used to rule out various haemoglobinopathies in subjects. Over the years, several well-established techniques have been developed with varying quantities and quality of DNA extracted and such procedures often involve tedious steps and cannot be used on large scale basis. Therefore, the endeavor of this study is to develop cost effective, with high quantity and quality of DNA that can be used for further molecular studies. In the present invention we have developed a method wherein sample requirement is less. This procedure may find application in laboratories with limited funded laboratories in India.
Background of the Invention
Haemoglobin disorders present a significant health problem in 71% of 229 countries, and these 71% of countries include 89% of all births worldwide. Over 3, 30,000 affected infants are born annually (83% sickle cell disorders, 17% thalassaemias). Haemoglobin disorders account for about 3.4% of deaths in children less than 5 years of age. Screening for haemoglobin disorders should form part of basic health services in most of the developing countries. In country like India where the basic needs are not fulfilled, peoples have to work day and night to get food for one time and they act as breadwinners. A home, in India where one time bread and butter are managed with great difficulty, if in that home a new born is suffering from haemoglobinopathies,

the condition will be miserable and that child will in mercy of God. Due to global migration j patterns, there has been an increase in the incidence of thalassaemia in many parts of the world. Various types of haemoglobinopathies common are beta- thalassaemia, alpha-thalassaemia and Sickle cell etc.
Beta-thalassaemia is inherited haemoglobin disorder characterized by absent ( β °) or decreased ( β ) synthesis of the β -globin chain. Thalassaemia is found in almost all populations and ethnic groups around the world. It has been estimated that 3% of the world's population of 150 million people carry 3-thalassaemia genes. Indeed, it is among the most common genetic diseases in the world. The severe forms of β -thalassaemia produce marked anaemia starting a few months after birth, and survival relies on regular blood transfusion and the lifelong use of drugs to prevent iron accumulation. β -Thalassaemias are among the commonest genetic disorders in the world, and more than 200 mutations have been described.
Alpha-Thalassaemia: results in a reduced rate of synthesis of alpha globin chain which is the main component of haemoglobin. Alpha thalassaemia can be classified into four types and severity of each typed is different depending on the number of the deletion of genetic loci for alpha globin. Alpha thalassaemia is commonly found in Southeast Asia, Southern China, India, Middle East and in the Mediterranean region. The two types of alpha thalassaemia, alpha thalassaemia and alpha thalassaemia carriers. The two alpha globin genes on the same chromosome are deleted in alpha thalassaemia. There is only one alpha globin gene deletion in an alpha- thalassaemia carrier. Moreover, some abnormal haemoglobin such as Hb Constant Spring (Hb CS) and Hb Pakse also affect the alpha globin gene expression and behave like and alpha thalassaemia. The interaction between these abnormal genes lead to many alpha thalassaemia syndromes such as homozygous alpha thalassaemia, Hb H disease (alpha thalassaemial/and alpha thalassaemia 2 or alpha thalassaemia 1/Hb CS) and Hb Bart's hydrops fetalis (homozygous alpha thalassaemial). Heterozygote screening and genetic counseling are essential for the prevention and control of severe thalassaemia diseases.
Traditional detection of thalassaemia and various haemoglobinopathies has relied heavily on the haematological tests. With the more generalized use of electronic cell counters, the diagnosis is first suspected by the discovery of a low mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) on routine "complete" blood counts. Increased levels of HbA2 (to 4-6 percent) and/or increased HbF (up to 5-20 percent) demonstrated by quantitative haemoglobin | electrophoresis supports the diagnosis. Unfortunately, the differential diagnosis between iron deficiency Anaemia and the β -thalassaemia trait can be difficult in practice, if there are not reciprocal increases in HbA2 and/or HbF. Moreover, in the presence of concomitant iron-

deficiency,HbA2 levels in" ( β -thaIassaemia individuals may fall into the normal range.
Occasionally, the diagnosis of iron deficiency cannot be made on the basis of measurements of
serum iron, iron binding capacity and/or the absence of stainable iron in the bone marrow. In
these instances, the demonstration of a reduced /5-globin synthetic rate (compared to a-globin).
generally employing H-leucine to analyze globin chain production in reticulocytes is required
for a conclusive diagnosis. This procedure is cumbersome and now well- suited for large scale
investigations.
In each type of haemoglobinopathies various types of mutation have been observed for example )
in case of beta-thalassaemia 200 mutation, to detect particular mutation PCR is best and reliable
molecular method employed for detection of various mutations in various haemoglobinopathies.
DNA of the effected subject is master key employed for an assortment of different molecular,
sophisticated research and for cutting edge technologies. The preference of DNA extraction
procedure requires an appraisal of several factors; the relative congruity of each factor is
determined by the setting under which the laboratory is operating. Some of the factors that need
i to be considered are costs, optimal yield of DNA, removal of substances that could influence the :
PCR reaction, the time and labour involved in the extraction procedure and maximum duration of
storage of the DNA without danger of contamination. The most suitable extraction procedure
should be financially and practically applicable in a low cost setting as well as deliver results that
are precise enough for interpretation in making diagnosis and help the clinicians to make right |
decision, in many methods the requirement of blood is 10ml for extraction of DNA used in PCR
and other studies (Malcolm, 1986; Old, et al. 2005 ).
Objects of the Invention
The main object of the present invention is to provide a method that can be used in low cost and
under moderate laboratory conditions. In this procedure the chemicals used are of low price and
are easy available.
It is another object of the invention to provide a process that can be used to isolate good quantity of DNA from the blood of the subject.
Statement of the Invention
Molecular analysis is the golden key for detection of various haemoglobinopathies in developing
countries. The extraction and isolation of DNA from blood is a necessary step for any PCR-based analysis. Hence, the method of DNA extraction is a key factor in determining the overall efficiency and reliability of any PCR-based test used to rule out various haemoglobinopathies in

subjects. Over the years, several well-established techniques have been developed with varying
quantities and quality of DNA extracted and such procedures often involve tedious steps and can
not be used on large scale basis. Therefore, the endeavor of this study is to develop cost effective.
with high quantity and quality of DNA that can be used for further studies. In the present
invention we have developed a method wherein sample requirement is less. This procedure may
find application in laboratories with limited funded laboratories in India. '
A summary of the Invention i
Accordingly, the present invention provides methodology for isolating the DNA from 1ml of
blood by using the chemicals and apparatus which are available in moderate settings. The blood of subject is taken with a consent form signed by that particular individual. The Blood is withdrawn under the ethical rules and regulation. The 1ml blood sample, centrifuge at 4000 rpm for 10 minutes at 4°C. The Supernatant was discard, to pellet add 1ml RBC lysate (incubate in ice for 10 minutes). After, 10 minutes mix gradually. Centrifuge at 4000 rpm for 10 minutes at 4°C, supernatant was discarded. Repeat 4-8 times till pellet appears white. To the white pellet (450 ul WBC lysate + 25 ul 10% SDS + 10 ul Proteinase K (10mg/ml).Mix till the pellet dissolves. Incubate at 37 °C for overnight (on water bath temp maintained at 37 °C).Add 500 μ1 equilibrated Phenol (Mix by vortexing gradually).Centrifuge at 4000rpm 10 minutes 4 °C. Take supernatant in fresh autoclave tube and add 500 μl of Chloroform iso-amlyalcohol (24:1).Mix by vortexing .Centrifuge at 4000rpm 10 minutes 4 °C. Take supernatant in fresh autoclave tube & 500ul of ice-chilled 100% ethanol (Mix manually by moving up and Down for 25 minutes).Centrifuge at 4000rpm for 10 minutes at 4°C. To the pellet add 500 Μ1 70 % ethanol. Centrifuge 4000 rpm 10 minutes at 4 °C. Supernatant was discarded. Repeat the process 3-6 times. Pellet was dried by placing in concentrator for 30 minutes. Add 100 ul lx TE buffer and incubated at 37 °C for 24-36 hours Store the DNA in freezer. The OD value is verified at 260nm and 280nm.
Detailed description of the invention with reference to examples
Thalassaemia is one of the major monogenic single gene disorders in the world population and it
was the first disease studied by using the techniques of molecular biology. Indian subcontinent , and South-East Asia have the highest prevalence of Beta-thalassaemia and comprise the so-called [ •thalassaemia belt.'A (Cao et al., 1997; Weatherall, et al., 2001). The inhabitant of India exhibits a wide range of genetic heterogeneity and, conservation and biological diversity-including the reservoir for occurrence of a large number of abnormal haemoglobin and thalassaemias in the world. Thalassaemia syndromes demonstrate that genetic information does

have more than a theoretical potential to have a major impact upon society. The thalassaemia syndromes were the first of human diseases to become painstakingly examined for the underlying molecular lesions by the application of molecular genetic strategies and recombinant DNA. Recently, with improvements in techniques in molecular biology and with the cloning of the human globin genes, cloning and sequencing the β globin genes from a patient with suspected thalassaemia (or the a globin gene from patients suspected to have alpha thalassaemia) have become possible. More recently, it has been found that a number of point mutations that result in the thalassaemic phenotype are genetically linked to specific restriction fragment length polymorphisms or haplotypes. Thus, by isolating DNA from peripheral tissues (usually leukocytes) , one can perform restriction enzyme digestion of genomic DNA and Southern blotting to examine for the presence of specific haplotypes from which one can infer the particular thalassaemic mutation. Master key of all the molecular study is DNA, which carries the genetic information from parents to offspring. The carrier of all the hereditary haemoglobinopathies is also the DNA. So for analysis and pedigree analysis the DNA is needed. The quantity and quality of DNA so be precise to perform the molecular analysis. The procedure employed to extract DNA from 1ml of blood is as under Example 1
DNA was extracted from peripheral EDTA anticoagulated whole blood: Draw 2 ml of blood using lavender-top Vacutainer (Beckton-Dickinson; EDTA anticoagulant). Keep cool until prep is performed (e.g. in an ice chest), but do not freeze. Highest yield will be achieved by extracting within 24 hours. 1ml blood sample is centrifuge at 4000 rpm for 10 minutes at 4°C .After certification supernatant was discarded. To the pellet, add 1ml RBC lysate (incubate in ice for 10 minutes). After 10 minutes, mix gradually and slowly. Centrifuge at 4000 rpm for 10 minutes at 4°C. Discard supernatant. Repeat the above processes 4 -8 times till the pellet will appear white. To the white pellet add 450 μl WBC lysate, 25 μl 10% SDS and 10 ul Proteinase K( 10mg/ml).Mix the contents properly till the pellet dissolves. Incubate at 37 °C for overnight (on water bath temp maintained at 37°C). After overnight incubation Add 500 ul equilibrated Phenol to the sample, mix by vortexing. Centrifuge at 4000rpm 10 minutes 4 °C . Take supernatant in fresh autoclave tube and add 500 μl of Chloroform iso-amlyalcohol (24:l).Mix by vortexing, Centrifuge at 4000rpm 10 minutes 4°C. Take supernatant in fresh autoclave tube & 500μl of ice-chilled 100% ethanol (Mix manually by moving the Eppendorf tube up and Down for 25 minutes).Centrifuge at 4000rpm for 10 minutes at 4°C. Discard the supernatant and to the pellet add 500 ul 70 % ethanol. Centrifuge 4000 rpm 10 minutes at 4 °C. Supernatant was discarded. Repeat the above process 3-6 times. Pellet was dried by placing in concentrator for 30 minutes.


'Add 100 μl 1x TE buffer and incubated at 37 °C for 24-36 hours. Store the DNA in freezer Example 2 Preparation of RBC Lysate:
I. Ammonium Bicarbonate, 0.072gm in 1000ml
II. Ammonium chloride, 7gm in 1000ml. (Sterilized by autoclaving)
Example 3
Preparation of WBC Lysate:
III. 0.5M EDTA (pH 8), 10ml in 200ml.
IV. NaCl, 0.876 gm in 1000ml (Sterilized by autoclaving)
Example 4
Preparation of 10% Sodium Dodecyl-Sulphate (SDS)
Example 5
Preparation of Equiliberated Phenol: The phenol is melted and to the melted phenol adds an
equal volume of buffer (0.5M Tris-Cl pH 8.0) at room temperature. Stir the mixture with
magnetic stirrer for 15 minutes. Turn off the stirrer and allow it to stand when two phase have
separated, aspirate as much as possible the upper (aqueous) phase using the glass pipette. Now
add an equal volume of 0.1M Tris-Cl (pH 8,0) to the phenol. Stir the mixture on the magnetic
stirrer for 15 minutes. Turn off the stirrers and when the two phases have separated, remove the
upper phase. Repeat the process till the pH of the phenolic phase is >7.8(measured with pH
paper).After phenol is equilibrated and the final aqueous phase has been removed, add 0.1
volume of 0.1M Tris-Cl (pH 8.0).
Example 6
Preparation of Chloroform Iso-amlyalcohol (24:1)
Example 7
Preparation of 70% of ethanol: 70ml of ethanol and rest volume with triple distilled water.
References:
1. Cao A, Luisella S, Galanello R, Rosatelli MC. Molecular diagnosis and carrier screening for p-thalassaemia. JAMA1997; 278:1273-7.
2. Malcolm S. Direct DNA analysis in family studies. Journal of Inherited Metabolic Disease.l986;9.
3. Old J., Traeger-Synodinos J. et al. Prevention of Thalassaemias and other haemoglobin disorders. Thalassaemia International ferderation publication.2005; 2:91.

I . n„ * „, _ „ ^ M rrmj_
4. Weatherall DJ, Clegg JB. The β-thalassaemia syndromes.4th Edn. Oxford: Blackwell Scientific publication; 2001; 635-87.

5- CLAIMS (not applicable for provisional specification. Claims should start with the preamble "I/we claim" on separate page)
We claim:
We claim:
1) DNA is extracted from 1ml of blood of the subject. In procedure the amount of sample required is less and it is cost effective (in developing country like India, where testing should be cost effective). The subjects suffering with thalassaemia have mild to severe anaemia. It becomes difficult to draw 5-10ml of blood for molecular and pathological tests.
2) According to claim 1 the 2ml of blood was taken from subject according to the ethical rules and regulation and 1 ml is used for DNA extraction and different chemicals were employed to extract the DNA. The chemicals were available in low price and easily available.
3) The Quantity and Quality of DNA is admirable to carry out the further molecular mechanism,