1

Isolation of DNA from Various Samples & Quantitative and Qualitative Analysis of DNA from Human Blood Stored At Different Temperature for Different Time Interval

Work carried out under the supervision of Dr. Preena Bhalla Department of Microbiology MAULANA AZAD MEDICAL COLLEGE Submitted By:SAPNA JAIN B M S III Yr.

Acknowledgment

A journey is easier when you travel together. Interdependence is certainly more valuable than independence. This project is the result of two months of work and observation, during which I have been guided, supported and helped in every possible aspect by many individuals. I take this opportunity to express my heartfelt gratitude to all of them. I am thankful to Dr. Preena Bhalla , Head of Department of Microbiology for her invaluable guidance. Her vast experience and insight into the subject has made things easier to comprehend. It was a matter of great privilege and pride to carry out this study under esteemed guidance of Dr. Pallavee, Scientist-III, and Department of Microbiology. I wish to express my sincere thanks and deep sense of gratitude for her guidance and unfailing help in every step of the study. Her constant supervision and encouragement enable me to complete the task. I am thankful to technical staff of Microbiology labs , for their constant help throughout the study. Last but not the least, I would like to put in my sincere gratitude to my internal guide Dr. Uma Chaudhary whose everlasting support, encouragement and love made me able to complete this work SAPNA JAIN

LIST OF ABBEREVIATION

bp CCD DNA C dNTP EDTA EtBr Fig g hr min mg ml mM MQ N

Base pairs charge coupled device Deoxyribonucleic acid Degree centigrade 2-deoxyribonucleotide 5-triphosphate Ethylene diamine tetra acetic acid Ethidium bromide Figure Gram hour minutes milligram milliliter millimolar Milli Q Water Normality

NaOH

Sodium hydroxide

iv

OD % RNA rpm SDS sec. TAE TBE TE Tris U UV V

Optical density Percentage ribose nucleic acid revolution per minutes Sodium dodecyl sulphate Seconds Tris acetate-EDTA Tris borate-EDTA Tris-EDTA Tris (hydroxymethyl) amino methane Units Ultraviolet Volts wavelegth

CONTENTS

Title Page Certificate Acknowledgement Abbreviations Table of Contents

i ii iii iv v

CHAPTER-

1. Introduction 2. Review Of Literature 3. Materials and Methods 4. Result & Conclusion 5. Appendices

1 8 17 21 23

INTRODUCTION

INTRODUCTION

Dna is the abbreviation of deoxy ribonucleic acid. It is a complex substance found in the nucleus of each cell and carries genetic information, which transmits from one generation to another. The dna of every individual with the exception of identical twins , I s unique. Avery, Macleod and Mccartey in 1944 described that dna is vehicle of generational transference of heritable unit. COMPONENETS OF DNA Dna is a polymer, its monomer are nucleotides and the polymer is known as polynucleotide. Each nucleotide consists of a 5-carbon sugar(deoxyribose),a nitrogen containing base attachted to the sugar and aphosphate group. There are four different types of nucleotides found in dna , only differing in their nitrogen bases.the four nucleotides are givenone letter abbreviation as shorthand for the four bases A for adenine C for cytosine G for guanine T for thymine

Purine bases Adenine and guanine are purines. Purines are the larger of the types of bases found in dna. Pyrimidine bases Cytosine and thiamine are pyrimidines. The 6 atoms( 4 carbon , 2 nitrogen) are numbered 1 to 6. Like purines all pyrimidines ring atoms lie n the same plane.

Deoxyribose sugar The doexyribose sugar forms the backbone, has 5 carbaon and 3 oxygen atoms. The carbon atoms are numbered 1,2,3,4,5 to distinguish from the numberings of the atoms of purines and pyridines ring. The hydroxyl group on the 5 and 3 carbon like to the phosphate groups to form thr dna backbone. Deoxyribose lacks a hydroxyl group at the 2 position when compared to ribose the sugar component of rna. Nucleoside A nucleoside is one of the four dna bases covalently attatched to the c1 position of a sugar.The sugar in deoxynucleosides is a 2 deoxy ribose. The sugar in ribonucleotides is ribose. Nucleosides differ from nucleotides as the lack phosphate group. The four different nucleosides are deoxyadenosine(da), deoxyguanosine(dg), deoxycytosine(dc), deoxythymidine(dt). Nucleotides A nucleotide is a nucleoside with one or more phosphate group covalently attached to the 3 or /and 5 hydroxyl group(s). Dna double helix Dna is normally a double stranded macromolecule. Two polynucleotide chains held with thermodynamic forces , forms a dna molecule. Two dna strands form a helical spiral,winding around a helix in a right handed spiral. Two polynucleotide shains run in opposite directions The sugar phosphate backbone of the dna helix wind around the helix axis like the railing of a spiral staircase.

10

The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of the spiral staircase.

Base pairs Within the dna double helix, a forms 2 binds with t on the opposite strand, and g forms 3 bonds with c on the opposite strands. Every strand of dna has pieces that contain genetic information which informs an organisms development(exons), and pieces that apparently supply no relevant genetic information at all(introns). Although the introns may seem useless, it has been found that they contain repeated sequences of base pairs. The sequences , called variable number of tandem repeats(VNTRs) can contain anywhere from 20 to 100 base pairs. In 1985,Prof. alec Jeffery, while studying the myoglobin gene, a protein that stores oxygen in muscle, discovered these regions of dna showing variation in number of tandem repeats. Hence name it as variable number of tandem repeats. It was discovered that the number of repeated sequences present in the sample could differ from one individual to another producing a unique pattern like finger print dictygraph and named it as dna finger prints. Dna finger prints are generally genetic pattern determined from an indiviuals genetic material i.e. dna. Tandemly repeated dna sequences are widespread throughout the human genome and show sufficient variability among individuals in a population, therefore have become important in several fields including genetic mapping, linkage analysis and human identity testing. In 1985, jeffreys developed a technique to study vntrs called the restriction fragment length polymorphism(rflp) and the technique of dna finger printing is often referred to as dna profiling. Presently this technique is extensively used for forensic purposes . Fingerprinting in the forensic sciences had generated considerable excitement in the criminal justice community. Earlier identity of an indiviual in forensic sciences was based on the ABO blood groups. But since 1970,other proteins and enzyme markers were used in the forensic evidences. But there was problem of low resolution,

Sources of DNA

1. Fresh blood 2. Blood stain

11

3. Semen and Seminal stains 4. Tissues(soft) 5. Bones(hard tissues) 6. Hair(roots) 7. Nails 8. Saliva

DNA FINGERPRINTING
Genetic, genomic, or DNA fingerprinting is the term applied to a range of techniques that are used to show similarities and dissimilarities between the DNA present in different individuals. Genetic fingerprinting is an important tool in the arsenal of forensic investigators. Genetic fingerprinting allows for positive identification, not only of body remains, but also of suspects in custody. Genetic fingerprinting can also link suspects to physical evidence. Sir Alec Jeffreys at the University of Leicester developed DNA fingerprinting in the mid 1980s. The sequence of nucleotides in DNA is similar to a fingerprint, in that it is unique to each person. DNA fingerprinting is used for identifying people, studying populations, and forensic investigations. DNA profiling (also called DNA testing, DNA typing, or genetic fingerprinting) is a technique employed by forensic scientist to assist in the identification of individuals on the basis of their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier. DNA profiling should not be confused with full genome sequencing. Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different to distinguish one individual from another. DNA profiling uses repetitive ("repeat") sequences that are highly variable, called Variable Number Of Tandem Repeats (VNTR). VNTRs locus are very similar between closely related humans, but so variable that unrelated individuals are extremely unlikely to have the same VNTRs.

APPLICATIONS DNA fingerprinting, a novel method to identify an individual has following has following applications:

12

1. Criminal cases (A) Rape (B) Murder (C) Kidnapping (D) Exchange of babies 2. Civil cases (A) Disputed paternity (B) Immigration (C) Identification of the nearest relation to claim compensation in mass disaster . 3. Population genetics (A) Animal genetics . (B) Plant genetics . (C) Clinical diagnosis .

Examples:1. Paternity and Maternity Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity. The patterns are so specific that a parental VNTR pattern can be reconstructed even if only the children's VNTR patterns are known (the more children produced, the more reliable the reconstruction). Parent-child VNTR pattern analysis has been used to solve standard father-identification cases as well as more complicated cases of confirming legal nationality and, in instances of adoption, biological parenthood. 2. Criminal Identification and Forensics DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence. VNTR patterns are also useful in establishing the identity of a homicide victim, either from DNA found as evidence or from the body itself. 3. Personal Identification The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the foreseeable future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is

13

both expensive and impractical. Social security numbers, picture ID, and other more mundane methods are much more likely to remain the prevalent ways to establish personal identification.

PROBLEMS Like nearly everything else in the scientific world, nothing about DNA fingerprinting is 100% assured. The term DNA fingerprint is, in one sense, a misnomer: it implies that, like a fingerprint, the VNTR pattern for a given person is utterly and completely unique to that person. Actually, all that a VNTR pattern can do is present a probability that the person in question is indeed the person to whom the VNTR pattern (of the child, the criminal evidence, or whatever else) belongs. Given, that probability might be 1 in 20 billion, which would indicate that the person can be reasonably matched with the DNA fingerprint; then again, that probability might only be 1 in 20, leaving a large amount of doubt regarding the specific identity of the VNTR pattern's owner. 1. Generating a High Probability The probability of a DNA fingerprint belonging to a specific person needs to be reasonably high--especially in criminal cases, where the association helps establish a suspect's guilt or innocence. Using certain rare VNTRs or combinations of VNTRs to create the VNTR pattern increases the probability that the two DNA samples do indeed match (as opposed to look alike, but not actually come from the same person) or correlate (in the case of parents and children). 2. Problems with Determining Probability A. Population Genetics B. Technical Difficulties Some Examples of DNA Uses for Forensic Identification

Identify potential suspects whose DNA may match evidence left at crime scenes Exonerate persons wrongly accused of crimes Identify crime and catastrophe victims Establish paternity and other family relationships Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers) Detect bacteria and other organisms that may pollute air, water, soil, and food Match organ donors with recipients in transplant programs Determine pedigree for seed or livestock breeds Authenticate consumables such as caviar and wine

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LITERATURE

15

EFFECT OF TEMPERATURE ON DNA EXTRACTED FROM FRESH BLOOD Whole blood is a common source of DNA, especially for genotype diagnostic
services. The effects of storage time (number of days from blood collection to DNA extraction) and temperature on DNA yield and quality are important. The temperature at which blood is stored and the time of storage affects the quantity and quality of the purified DNA. For short-term blood storage (<5 days), blood should be collected in EDTA tubes and stored at 4C to obtain maximum DNA yield. For long-term storage, blood should be frozen at -80C.

Agarose Gel Electrophoresis

Introduction: Agarose gel electrophoresis is a widely used method that separates molecules based upon charge, size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and proteins. Agarose gel electrophoresis possesses great resolving power, yet is relatively simple and straightforward to perform. The gel is made by dissolving agarose powder in boiling buffer solution. The solution is then cooled to approximately 55oC and poured into a mold where it solidifies. The gel is submerged in a buffer-filled chamber, which contains electrodes. Samples are prepared for electrophoresis by mixing them with components that will give the mixture density, such as glycerol or sucrose. This makes the sample denser than the electrophoresis buffer. These samples can then be loaded with a micropipet or transfer pipet into wells that were created in the gel by a template during casting. The dense samples sink through the buffer and remain in the wells. A direct current power supply is connected to the electrophoresis apparatus and current is applied. Charged molecules in the sample enter the gel through the walls of the wells. Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode (cathode). Within a range, the higher the applied voltage, the faster the samples migrate. The buffer serves as a conductor of electricity and to control the pH. The pH is important to the charge and stability of biological molecules. Agarose is a polysaccharide derivative of agar. It contains microscopic pores, which act as a molecular sieve. The sieving properties of the gel influence the rate at which a molecule migrates. Smaller molecules move through the pores more easily than larger ones. Molecules

16

can have the same molecular weight and charge but different shapes. Molecules having a more compact shape (a sphere is more compact than a rod) can move more easily through the pores. Factors such as charge, size and shape, together with buffer conditions, gel concentrations and voltage, affect the mobility of molecules in gels. Given two molecules of the same molecular weight and shape, the one with the greater amount of charge will migrate faster. In addition, different molecules can interact with agarose to varying degrees. Molecules that bind more strongly to the agarose will migrate more slowly.

UV VISIBLE SPECTROPHOTOMETRTY

Uv visible spectrophotometry/ spectroscopy involves the spectroscopy of protons and spectrophotometry. It uses the light in the visible range and adjacent near the ultraviolet (UV) and infrared ranges. In this region of energy space molecules undergo electronic transitions. This technique is routinely used in quantitative determination of solutions of transition metal ions and highly conjugated organic compounds. Solutions of transition metal ions can be coloured(i.e. absorb visisble light( because the electrons within the metal ions can be excited from one electronic state to other.the colour of the metal ion solution is strongly affected by the prescence of other species like ligand. For instance,the colour of dilute copper sulphate solution is very light blue, adding ammonia intensifies the colur and changes the wavelength of maximum adsorption( max)

Organic compounds , especially those with a high degree of conjugation , also absorb light of the uv or visible region of electromagnetic spectrum. The solvents for these determinations are often water for water soluble compounds or ethanol for organicsoluble compounds. While charge transfer complexes also gives rise to colour .the colours are often too intensive to be used for quantitative measurement. The beer-lambert law states that absorbance of a solution is directly proportional to its concentration. The uv-visible spectroscopy is used to determine the concentration of a solution. It is necessary to know how quickly absorbance changes with the concentration.this can be determined froma calibration curve. A uv-visible spectroscopy may be used as a detector for hplc. The prescence of an analyte that gives a response which can be assumed to be proportional to the concentrataion. The response ( peak)for a particular concentration is known as the response factor. For accurate response compare the response of unknown to the response of standard( same a s calibration curves.

17

The instrument used in the uv-visible spectroscopy is called uv- visible spectrophotometer. It measures the intensity of light passing through a asample(I), and compares it to the intensity of light before passing through the sample(IO). thr ratio I/ IO is called the transmittance and expressed as percentage(%T). the absorbance A is based on the transmittance, A=-log(%T).

The basic parts of a spectrophotometer are a light source(often an incandescent bulb in visible range and a deuterium lamp in the uv range) , a sample holder, a diffraction grating or monochromator to separate different wavelengths of light and a detector. The detector is generally a photodiode or a CCD. Photodiodes a re used with monochromators, which filter the light so that onl light of a single wavelength reaches the detector. Diffraction gratings are used with CCDs which collects light of different wavelengths on different pixels. A spectrophotometer can be either single beam or double beam. In a single beam instrument, all the light passes through the sample cell, IO must be measured by removing the sample. This was the earliest design , but is still in common use in teaching and industries. In double beam instrument , light splits into two beams before it reaches the sample. One beam is used as the reference and the other passes through the sample. Some of these instruments have two detectors , so reference and sample are measured at the same time. In other instruments , two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and reference beam. Sample of this spectrophotomety are generall liquid, although absorbance of gases and solid can also be measured. The samples are placed in the transparent cells called cuvettes. Cuvettes are typically rectangular in shape, commonly with an internal width of 1 cm( this width acts a s path length in lamberts- beer law). Good quality cuvettes are generally made of high quality quartz, although glass or plastic cuvettes are common( glas and plastic absorb uv range therefore affecting the accuracy of results).

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MATERIAL REQUIREMENTS

19

CHEMICALS:

Serial no. 1. 2.

Chemicals

Procurement

Agarose Alcohol

SISCO Research Lab, Mumbai Merck Company Qualigen SISCO Qualigen Qualigen Mumbai SISCO Research Lab, Mumbai Merck Qualigen Merck Qualigen Merck Qualigen Qualigen Qualigen Qualigen SISCO Research Lab, Mumbai Biometer Research Lab,

Serial No. Boric Acid Enzyme 3. 1. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Proteinase K Choroform E.D.T.A Ethidium Bromide Formaldehyde Glacial Acetic Acid Hydrochloric Acid Isoamyl Alcohol Magnesium Chloride Phenol Propanol Sodium Acetate SDS Tris Buffer Milli Q

Enzymes used:

20

Serial no. 1. 2. 3. 4. 5.

Glassware Beaker Conical Flask Glass plate Glass Pipettes Measuring cylinder

Company Borosil, India Borosil, India Borosil, India Borosil, India Borosil, India

Glassware Used

Equipment used:

21

Serial No. 1. 2. 3. 4. 5 6. 7. 8. 9. 10. 11. 12. 13. 14.

Equipment Used Autoclave Shaking Water Bath Oven Microwave oven Refrigated centrifuge Micropipette Deep frezzer (-80C) Freeze (-20C to 4C) PCR centrifuge Agarose gel apparatus PAGE Apparatus Gel doc. Electronic Balance Thermal cycler

Company Yorko Scientific industry Nirmal international Thermotech BPL Heraeus Multifuge 3S-R Brand Thermo Forma Whirlpool Refrigrator Napco Thermo Electron Corporation Biometra Alpha innotech corporation Denver instrument APX-200 Peltier Thermal cycler (PTC200)

15. 16

Magnetic stirrer Vortex

Spinot Yorko Scientific Industry

Plastic Ware Used

22

Serial no. 1. 2. 3.

Plastic Ware Centrifuge tube micropipette Micropipette Tips

Company Tarson Tarson Tarson

23

METHOD

24

METHOD Procedure summary 1. Sample collection and preservation. 2. The DNA was extracted from various samples and analyzed quantitatively and qualitatively. 3. Then DNA extracted from whole fresh blood was stored at different temperature for a particular period of time, then it was analyzed quantitatively and qualitatively by Agarose gel electrophoresis.

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SAMPLE COLLECTION
Whole blood sample was taken and kept in 1.8% EDTA solution and stored.

DNA EXTRACTION FROM VARIOUS SAMPLES

METHOD OF DNA EXTRACTION FROM BLOOD 1. Take 500l of blood sample and add equal volume (500l) lysis buffer-1 in a centrifuge tube. 2. Keep it at - 80C for 2 hrs.
3. Now incubate the solution at 60C for 10 min

4. Centrifuge the tube for 15 min at 4600 rpm. 5. Discard the supernatant. 6. Take the pellet and add 500l of lysis buffer-2. 7. Dissolve the pellet in lysis buffer-2 and add SDS (2% volume of blood sample). 8. Add 5l of proteinase K.
9. Vortex the tube to dissolve the pellet in lysis buffer and incubate the sample for

overnight at 37C in water bath (atleast 14-16 hrs).

26

10. Add equal volume of saturated phenol (pH 8). 11. Mix it for 15 min on rotospin and then centrifuge it for 15 min at 4600 rpm at 4C. 12. Two liquid layers will developed, upper one is aqueous containing the DNA and the lower phase is the organic phase. 13. Take the aqueous phase in fresh centrifuge tube with the help of sterile pipette 14. Add equal volume of phenol and chloroform in the ratio of 1:1. 15. Mix it for 15 min on rotospin and centrifuge at 4600 rpm at 4C. 16. Separate the aqueous phase in another fresh centrifuge tube and add equal volume of chloroform and iso-amyl alcohol in the ratio of 24:1. 17. Mix well for 15 min on rotospin and then centrifuge for 15 min at 4600 rpm at 4C. 18. Take the aqueous phase in fresh centrifuge tube. 19. Add 1/10 of sodium acetate and chilled propanol, it will precipitate the DNA,. 20. Give it a pop spin for obtaining tact form of DNA.
21. Centrifuge the tube at 4600 rpm for 5 min at 4C.

22. After centrifugation DNA settled to the pellet, discard the whole aqueous phase carefully.
23. For washing of DNA, add 500l of 70% alcohol and centrifuge it for 5 min at 4600

rpm, at 4C (repeat this step twice).

27 24. Discard the 70% ethanol and leave the DNA for drying at room temperature.

25. Dissolve the DNA in 500 l of TE buffer. 26. The DNA sample obtained was ready for further processing.

1.

METHOD OF DNA EXTRECTION FROM SALIVA Take 500l of blood sample and add equal volume (500l) lysis buffer in a

centrifuge tube. 2. 3. 4. 5.
6.

Add 50l SDS (2% ) Add 5l of proteinase K. Incubate the sample for overnight at 37C in water bath (atleast 14-16 hrs). Add equal volume of saturated phenol (pH 8). Mix it for 15 min on rotospin and then centrifuge it for 15 min at 4600 rpm at 4C. Two liquid layers will developed, upper one is aqueous containing the DNA and the

7.

lower phase is the organic phase. 8. 9. 10. Take the aqueous phase in fresh centrifuge tube with the help of sterile pipette Add equal volume of phenol and chloroform in the ratio of 1:1. Mix it for 15 min on rotospin and centrifuge at 4600 rpm at 4C.

28

11.

Separate the aqueous phase in another fresh centrifuge tube and add equal volume of

chloroform and iso-amyl alcohol in the ratio of 24:1. 12. 13. 14. 15.
16.

Mix well for 15 min on rotospin and then centrifuge for 15 min at 4600 rpm at 4C. Take the aqueous phase in fresh centrifuge tube . Add 1/10 of sodium acetate and chilled propanol, it will precipitate the DNA,. Give it a pop spin for obtaining tact form of DNA. Centrifuge the tube at 4600 rpm for 5 min at 4C. After centrifugation DNA settled to the pellet, discard the whole aqueous phase

17.

carefully.
18.

For washing of DNA, add 500l of 70% alcohol and centrifuge it for 5 min at 4600

rpm, at 4C (repeat this step twice).

19.

Discard the 70% ethanol and leave the DNA for drying at room temperature. Dissolve the DNA in 500l of TE buffer. The DNA sample obtained was ready for further processing.

20. 21.

1.
2.

METHOD OF DNA EXTRECTION FROM TISSUE Take 1g of tissue and cush it with the help of tissue homogenization. Add 50l of SDS (2%).

29

3. 4. 5. 6.
7.

Add 5l of proteinase K. Incubate the sample for overnight at 37C in water bath (atleast 14-16 hrs). Add equal volume of saturated phenol (pH 8). Mix it for 15 min on rotospin and then centrifuge it for 15 min at 4600 rpm at 4C. Two liquid layers will developed, upper one is aqueous containing the DNA and the

lower phase is the organic phase. 8. 9. 10. 11. Take the aqueous phase in fresh centrifuge tube with the help of sterile pipette Add equal volume of phenol and chloroform in the ratio of 1:1. Mix it for 15 min on rotospin and centrifuge at 4600 rpm at 4C. Separate the aqueous phase in another fresh centrifuge tube and add equal volume of

chloroform and iso-amyl alcohol in the ratio of 24:1.

12.

Mix well for 15 min on rotospin and then centrifuge for 15 min at 4600 rpm at 4C. Take the aqueous phase in fresh centrifuge tube . Add 1/10 of sodium acetate and chilled propanol, it will precipitate the DNA,. Give it a pop spin for obtaining tact form of DNA. Centrifuge the tube at 4600 rpm for 5 min at 4C. After centrifugation DNA settled to the pellet, discard the whole aqueous phase

13. 14. 15.

16. 17.

carefully.

30 18.

For washing of DNA, add 500l of 70% alcohol and centrifuge it for 5 min at 4600

rpm, at 4C (repeat this step twice).

19.

Discard the 70% ethanol and leave the DNA for drying at room temperature. Dissolve the DNA in 500 l of TE buffer. The DNA sample obtained was ready for further processing.

20. 21.

1.

METHOD OF DNA EXTRACTION FROM BLOOD STAIN Scrap the blood stain and collect the stain in 15ml tarsons add equal volume (500l)

forensic buffer . 2. 3. 4. 5. 6. 7. 8. 9. Incubate it at 37C for 3-4 hrs in a waterbath. Centrifuge the tube for 15 min at 4600 rpm. Wash twice with PBS-Phosphate buffer saline to remove the dirt. Discard the supernatant. Take the pellet and add 500l of forensic buffer. Dissolve the pellet in forensic buffer and add 50l SDS . Add 5l of proteinase K. Vortex the tube to dissolve the pellet in lysis buffer and incubate the sample for

overnight at 37C in water bath (atleast 14-16 hrs).

31

10.
11.

Add equal volume of saturated phenol (pH 8). Mix it for 15 min on rotospin and then centrifuge it for 15 min at 4600 rpm at 4C. Two liquid layers will developed, upper one is aqueous containing the DNA and the

12.

lower phase is the organic phase. 13. 14. 15. 16. Take the aqueous phase in fresh centrifuge tube with the help of sterile pipette Add equal volume of phenol and chloroform in the ratio of 1:1. Mix it for 15 min on rotospin and centrifuge at 4600 rpm at 4C. Separate the aqueous phase in another fresh centrifuge tube and add equal volume of

chloroform and iso-amyl alcohol in the ratio of 24:1. 17. 18. 19. 20.
21.

Mix well for 15 min on rotospin and then centrifuge for 15 min at 4600 rpm at 4C. Take the aqueous phase in fresh centrifuge tube . Add 1/10 of sodium acetate and chilled propanol, it will precipitate the DNA,. Give it a pop spin for obtaining tact form of DNA. Centrifuge the tube at 4600 rpm for 5 min at 4C. After centrifugation DNA settled to the pellet, discard the whole aqueous phase

22.

carefully.
23.

For washing of DNA, add 500l of 70% alcohol and centrifuge it for 5 min at 4600

rpm, at 4C (repeat this step twice).

32 24.

Discard the 70% ethanol and leave the DNA for drying at room temperature. Dissolve the DNA in 500 l of TE buffer. The DNA sample obtained was ready for further processing.

25. 26.

1.

METHOD OF DNA EXTRECTION FROM SEMEN Take 500l of semen sample and add equal volume (500l) seminal buffer in a

centrifuge tube. 2. 3. 4. Add 50l of DTT(Dithiothreitol) Add 5l of proteinase K. Vortex the tube to dissolve the pellet in lysis buffer and incubate the sample for

overnight at 37C in water bath (atleast 14-16 hrs). 5.

6.

Add equal volume of saturated phenol (pH 8). Mix it for 15 min on rotospin and then centrifuge it for 15 min at 4600 rpm at 4C. Two liquid layers will developed, upper one is aqueous containing the DNA and the

7.

lower phase is the organic phase. 8. 9. 10. Take the aqueous phase in fresh centrifuge tube with the help of sterile pipette Add equal volume of phenol and chloroform in the ratio of 1:1. Mix it for 15 min on rotospin and centrifuge at 4600 rpm at 4C.

33

11.

Separate the aqueous phase in another fresh centrifuge tube and add equal volume of

chloroform and iso-amyl alcohol in the ratio of 24:1. 12. 13. 14. 15.
16.

Mix well for 15 min on rotospin and then centrifuge for 15 min at 4600 rpm at 4C. Take the aqueous phase in fresh centrifuge tube . Add 1/10 of sodium acetate and chilled propanol, it will precipitate the DNA,. Give it a pop spin for obtaining tact form of DNA. Centrifuge the tube at 4600 rpm for 5 min at 4C. After centrifugation DNA settled to the pellet, discard the whole aqueous phase

17.

carefully.
18.

For washing of DNA, add 500l of 70% alcohol and centrifuge it for 5 min at 4600

rpm, at 4C (repeat this step twice).

19.

Discard the 70% ethanol and leave the DNA for drying at room temperature. Dissolve the DNA in 500 l of TE buffer. The DNA sample obtained was ready for further processing.

20. 21.

1.

METHOD OF DNA EXTRECTION FROM HAIR FOLLICLE Take 25 hair follicles and add equal volume (500l) hair lysis buffer in a centrifuge

tube.

34

2. 3. 4.

Add 50l of SDS. Add 5l of proteinase K. Vortex the tube to dissolve the pellet in lysis buffer and incubate the sample for

overnight at 37C in water bath (atleast 14-16 hrs). 5.

6.

Add equal volume of saturated phenol (pH 8). Mix it for 15 min on rotospin and then centrifuge it for 15 min at 4600 rpm at 4C. Two liquid layers will developed, upper one is aqueous containing the DNA and the

7.

lower phase is the organic phase. 8. 9. 10. 11. Take the aqueous phase in fresh centrifuge tube with the help of sterile pipette Add equal volume of phenol and chloroform in the ratio of 1:1. Mix it for 15 min on rotospin and centrifuge at 4600 rpm at 4C. Separate the aqueous phase in another fresh centrifuge tube and add equal volume of

chloroform and iso-amyl alcohol in the ratio of 24:1. 12. 13. 14. 15.
16.

Mix well for 15 min on rotospin and then centrifuge for 15 min at 4600 rpm at 4C. Take the aqueous phase in fresh centrifuge tube . Add 1/10 of sodium acetate and chilled propanol, it will precipitate the DNA,. Give it a pop spin for obtaining tact form of DNA. Centrifuge the tube at 4600 rpm for 5 min at 4C.

35

17.

After centrifugation DNA settled to the pellet, discard the whole aqueous phase

carefully.
18.

For washing of DNA, add 500l of 70% alcohol and centrifuge it for 5 min at 4600

rpm, at 4C (repeat this step twice).

19.

Discard the 70% ethanol and leave the DNA for drying at room temperature. Dissolve the DNA in 500 l of TE buffer. The DNA sample obtained was ready for further processing.

20. 21.

1.

METHOD OF DNA EXTRECTION FROM TOOTH

PRETREATMENT

Wash the tooth sample in 0.1 Hypochloride solution Then twice with Milli Q. Add equal volume (500l) extraction buffer-I in a centrifuge tube. Add 50l of SDS . 4. Add 5l of proteinase K. 5. Vortex the tube to dissolve the pellet in lysis buffer and incubate the sample for overnight at 37C in water bath (atleast 14-16 hrs).
6. Add equal volume of saturated phenol (pH 8).

2. 3.

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7. Mix it for 15 min on rotospin and then centrifuge it for 15 min at 4600 rpm at 4C. 8. Two liquid layers will developed, upper one is aqueous containing the DNA and the lower phase is the organic phase. 9. Take the aqueous phase in fresh centrifuge tube with the help of sterile pipette 10. Add equal volume of phenol and chloroform in the ratio of 1:1. 11. Mix it for 15 min on rotospin and centrifuge at 4600 rpm at 4C. 12. Separate the aqueous phase in another fresh centrifuge tube and add equal volume of chloroform and iso-amyl alcohol in the ratio of 24:1. 13. Mix well for 15 min on rotospin and then centrifuge for 15 min at 4600 rpm at 4C. 14. Take the aqueous phase in fresh centrifuge tube . 15. Add 1/10 of sodium acetate and chilled propanol, it will precipitate the DNA,. 16. Give it a pop spin for obtaining tact form of DNA.
17. Centrifuge the tube at 4600 rpm for 5 min at 4C.

18. After centrifugation DNA settled to the pellet, discard the whole aqueous phase carefully.
19. For washing of DNA, add 500l of 70% alcohol and centrifuge it for 5 min at 4600

rpm, at 4C (repeat this step twice).

20. Discard the 70% ethanol and leave the DNA for drying at room temperature.

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21. Dissolve the DNA in 500 l of TE buffer. 22. The DNA sample obtained was ready for further processing.

AGROSE GEL ELECTROPHORESIS OF DNA :

Gel electrophoresis is a method that separates macromolecules either nucleic acids or proteins on the basis of size, electric charge and other chemical properties. A gel is a colloid in solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to energy of an electric field. Phoresis, from the Greek verb phoros means to carry across. Thus, gel electrophoresis refers to the technique in which molecules are forced across a spin of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. Molecular properties determine how rapidly an electric field can move the molecule through a gelatinous medium. Many important biological molecules such as amino acid, posses ionisable group and, therefore at any given pH exist in solution as electrically charged species either as cations (+) or anions (-) depending on the cathode or to the anode. The following factors should be considered for the rate at which DNA fragment migrate in agarose : 1. Agarose concentration should be 0.8%.

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2. The size of DNA fragment- linear double stranded molecule travel through the gel matrix at the rate inversely proportional to the log of their molecular weight. Smaller is the fragment faster it moves. 3. DNA conformation DNA shape also influence the migration rate for the DNA fragment with a particular size, the migration rate will be: Closed circular > Super coiled > linear > open circular. i. Current applied - the migration of the DNA is directly proportional to the voltage applied, too high to low voltage is not recommended. The normal voltage should be 60 to 70. The distance is referred to as the length to the gel between negative and positive electrode. DNA gels are generally stained with 1 micro gram/ml ethidium bromide, prepared under short/medium wave UV trans-illuminator. Molecular marks DNA runs along with sample DNA to detect the fragment size as the standard. DNA samples are loaded with the bromophenol blue. So the position of DNA can be localized during electrophoresis.

ii.

Agarose Gel Electrophoresis is carried out to ensure the presence and quality of DNA. 1. 0.8% (800mg/100ml) of agarose powder was weighed and dissolved in 1x TBE buffer, in a conical flask and heat the slurry in microwave oven until the agarose dissolved. 2. After this, solution was cooled to ~55C, 5l of EtBr was added and the contents of the flask were mixed well. 3. A square toothcomb was placed in the gel box (plastic gel tray with rubber detachments at two ends) and positioned 0.5 1.0mm above the base of the tray, to forms wells. 4. Then the agarose solution was poured into the gel casting tray and allowed to solidify (30 min).

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5. After solidification of the gel, the combs were removed and then gel tray was placed in 1x TBE buffer in buffer tank such that it was fully submerged in buffer. 6. DNA sample (3l) were mixed with loading dye-bromophenol blue (2l) and carefully loaded into each wells. 7. The lid to the buffer tank was closed and electrodes were plugged into a power source and a voltage of 1-5 v/cm (measured as the distance between the positive and negative electrode) was applied. 8. When the dye and DNA samples had migrated a sufficient distance through the gel the electric current was turned off and the leads were removed. 9. The gel was examined under the UV light in the UV imager (Alfa imager). The presence of DNA was justified by the fluorescing bands observed (as a result of DNAEtBr intercalation).

Quantification of DNA

The quantification of DNA is done to the calculate the quality and quantity of DNA obtained from the tissue sample for further analysis. This is done by UV spectrophotometer. Formula: Quality of DNA is sample = O.D. at 260nm / O.D. at 280nm. The ratio will give the quality of DNA ranging from 1.8 to 2.0. Quantity of DNA in the sample = O.D. at 260nm x 50 x dilution

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DIFFERENT TEMPERATURE AND STORAGE TIME FOR DNA BEFORE EXTRACTION

1. 6 ml Whole blood was taken from me and 1.8%EDTA was added 2. It was divided into 4 partsa) 500l for 0 week isolation b) 1500 l for 1st week isolation c) 1500 l for 2nd week isolation d) 1500 l for 3rd week isolation 3. In each week we divide the whole blood in 3 parts, each kept at 4C, RT (room temperature),-80C respectively. 4. Then DNA is extracted week after week and stored. 5. Now DNA is analyzed quantitatively and qualitatively by agarose gel and spectrophotometer.

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RESULT

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DNA EXTRACTED FROM VARIOUS SAMPLES

I.

Agarose gel electrophoresis (Qualitative analysis)

Agarose Gel under UV light Where:sample no. 1 2 3 4 5 6 7 sample blood saliva brain tissue semen blood stain hair folicle tooth

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II.

Nano drop spectrophotometer(Quantitative analysis)

S No. 1. 2. 3. 4. 5. 6. 7.

Sample Blood Saliva Brain Tissue Semen Blood Stain Hair Follicle Tooth

O.D. at 260 nm 1.144 3.312 2.551 3.812 5.360 3.322 5.792

O.D. at 280 nm 0.629 2.098 1.430 2.242 3.425 1.965 3.367

Ratio=A260/ A280 1.82 1.60 1.78 1.70 1.65 1.69 1.72

Conc. (ng/ul) 150.6 166.6 1297.6 165.6 281.5 166.5 289.6

NOTE:-The DNA is considered to be of good quality when the ratio comes out to be between 1.6-1.8

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DNA EXTRACTED FROM WHOLE BLOOD AND STORED AT DIFFERENT TEMPERATURE FOR 7,14,21 DAYS

I.

Agarose gel electrophoresis (Qualitative analysis)

Agarose Gel under UV light

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Where:-

sample no. 1 2 3 4 5 6 7 8 9 10 11 12

sample negative control positive control fresh blood (0 week) -80 c-1st week 4 c - 1st week rt- 1st week -80 c -2nd week 4 c- 2nd week rt- 2nd week -80 c-3rd week 4 c - 3rd week rt- 3rd week

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III.

Nano drop spectrophotometer(Quantitative analysis)

S No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Sample 0 week -80 c(1stweek) 4 c(1st week) RT(1st week) -80 C(2ndweek) 4 C(2ndweek) RT(2ndweek) -80 C(3rdweek) 4 C(3rdweek) RT(3rdweek)

O.D. at 260 nm 8.749 7.334 6.026 6.449 7.327 5.826 6.336 6.726 5.593 5.943

O.D. at 280 nm 4.859 4.177 3.385 3.793 4.064 3.301 3.863 3.779 3.196 3.396

Ratio=A260 /A280 1.80 1.76 1.78 1.70 1.80 1.76 1.72 1.78 1.75 1.75

Conc. (ng/ul) 437.46 366.70 301.34 322.45 356.33 291.32 316.80 336.34 279.67 297.15

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CONCLUSION

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We compared DNA yield and quality from bovine blood samples exposed to storage time (7, 14 or 21 days) and temperature (-80 degrees, 4 degrees, 37 degrees C) to the results obtained when DNA was extracted within 4 h of collection. The highest mean DNA yields, relative to the control samples, were obtained from blood stored at 4 degrees C. Blood stored at 37 degrees C for > or = 3 days or 23 degrees C for > or = 7 days yielded less (P < .05) DNA, compared with blood stored at 4 degrees or -20 degrees C for up to 28 days. The genomic DNA obtained was of high molecular weight and suitable for restriction enzyme digestion and PCR amplification, regardless of the treatment.

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50

APPENDIX

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Preparation of Reagent Used

A)

Lysis Buffer-1 : 30mM Tris 5mM EDTA 50mM NaCl 3.6436gm. Tris, 1.861gm. EDTA, 2.922gm NACl dissolve in 1 liter of Milli Q with the help of magnetic stirrer, and then autoclaved. The autoclaved solution is kept at 4C. Function : to lyse the RBC.

B)

Lysis Buffer-2 : 75mM NaCl 20mM EDTA 4.373gm. NACl, 0.744gm EDTA dissolve in liter of Milli Q with the help of magnetic stirrer, and then autoclaved. The autoclaved solution is kept at 4C. Function : to lyse WBC.

C)

SDS(20%) : 200gm SDS dissolved in 1000ml of Milli Q by gentle mixing. Function : it is the detergent, which lyses the cell by removing lipd molecules and thereby causing the disruption of the cell membrane.

D)

Proteinase K : 20 mg per ml of milli Q. Source : Mould Tritirachium album Function : it breaks the cell wall proteins into smaller units.

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E)

Saturation of phenol : Melt the phenol crystals at 68C in the water bath. Add 0.5gm hydroxyquinoline per 100 ml of phenol. This acts as an oxidant. Add equal volume of 0.5M Tris HCl. Mix well for 15 minutes. Keep at room temperature till 2 layers separate out. Discard the aqueous layer. Add and equal amount of tris HCl. Repeat the process till the pH comes above 7.8 Add 0.2% mercapto ethanol containing tris to it. This helps to protect it from

oxidation. Function : phenol washing is carried out to deproteinise the cell extract. The organic solvent precipitates the protein but leaves the nucleic acids (DNA & RNA) in aqueous solution.
F)

Cholorform & iso-amyl alcohol (24:1): 2ml Iso-amyl alcohol and 48 ml

Chloroform to be mixed and used when required. Function: same purpose as phenol for purification of nucleic acids from protein. It may also play a role in the removal of RNA.
G)

Sodium acetate and chilled Propanol : the salt solution is used to precipitate the

DNA into a strand, which is visible to the naked eye.

H)

70% ethanol : 70 ml of absolute alcohol in 30 ml of Milli Q. Function : it is used to wash the DNA.

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OTHER REAGENTS:I)

5M EDTA (pH-8.0) : 186.12gm of EDTA dissolved in 800ml of milliQ with the

help of magnetic stirrer, now adjust the pH 8.0 with NaOH then make up the volume 1000ml with miliQ and stored at 4C.
J)

Tris HCl (pH-8.0) : 30gm Tris dissolved in 500ml milliQ with the help of magnetic

stirrer then check the pH(approximate >9.0) to maintain the pH 8.0 with help of HCl.
K)

TBE 10x : 108.0gm Tris Base, 55.0gm Boric acid, and 40ml 0.5M EDTA dissolved

in 1000ml of milliQ.
L)

Ethidium Bromide : 10mg Ethidium Bromide dissolved in 1ml of milliQ. FUNCTION : it is used to visualize DNA on the gel.

M)

Loading Dye (Bromophenol Blue) : sucrose 40%, Bromophenl blue 0.25%, xylene

0.25% and dissolved in milliQ. FUNCTION : Used to load the DNA in the well and check its migration on the gel.
N)

Solution for Agarose gel electrophoresis : 1X TBE (pH 8) for 50 ml. 5ml 10X TBE buffer mixed in 45ml MilliQ 0.8 % AGAROSE GEL : 0.8 gm agarose per 100ml.

0.8 gm agarose dissolved in 10 ml 10X TBE + 90 ml MilliQ in conical flask. This solution is boiled in the microwavw oven till it becomes transparent. The solution was cooled to

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60C and added 5l ethidium bromide dye. Now the gel is ready for casting in the casting tray.

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PRECAUTION 1. Blood should be handled with appropriate precautions to avoid exposure to infectious agents. 2. EDTA as an anticoagulant is preferred. 1 week old blood gives less yield unless the have been stored frozen. 3. Disposable gloves should be used 4. Reagents should be freshly made or autoclaved, as appropriate .
5. Layer separation should be done carefully.

6. Ethidium bromide should be handled from a distance. 7. Layers of phenol should not be mixed. 8. Balance should be maintained in the centrifuge. 9. Apparatus for gel should be cleaned properly.