Dna Fingerprinting

DNA FINGERPRINTING Seminar Report
CHAPTER -1
INTRODUCTION
Oilseed rape (OSR) (Brassica nap us L.) is one of the major oilseed crops grown in
Europewith over 3.5 million hectares under cultivation in the EU in 1999. OSR is primarily
grownfor edible oil extraction. However, the process also generates a large quantity of de-fatted
meal (around 1.5million ton in the UK) which is mainly used as a low-value additive to animal
feeds. The recent increasing production of biodiesel from OSR in Western Europe (around 2
million tons anticipated in 2003) is also expected to increase the meal production as a by-
product.
The opportunities for increasing use of OSR meal in animal feed are limited bythe
presence of glucosinolates, which impose limits on inclusion rates in diets for poultry and pork
production. There are further difficulties in the utilisation of the meal from industrial oil crops
such as high erucic acid rapeseed (HEAR) due to the presence of high levels of glucosinolates;
that have been reduced by breeding in conventional '00' OSR crops.
The de-oiled OSR meal is a rich source of structural and bioactive proteins with a range
ofExploitable properties that are only just starting to be realised. A higher profitability of the
rapeseed integrated chain would only be achieved by developing new higher-value applications
for these protein fractions. Some recent results highlight the feasibility of protein extraction
processes, and the economic potential of the resulting innovative protein products, especially in
the non-feed area (Sanchez-Viroqua et al., 2001, Gerbanowski ET al.2003).
During the last 20 years, the breeding objectives for the seed quality of OSR have
beenmainly concerned with the quantitative and qualitative improvement of the oil fraction and a
decrease in the content of glucosinolates in meal.it is looks likely that OSR will be grown and
used in the future for many non-food applications.
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CHAPTER-2
GENETIC VARIABILITY
2.1 DNA fingerprinting:

DNA fingerprinting techniques were assessed for the estimation of genetic variability
within an OSR cultivar, and between different cultivars. The methods tested were based on
determination of microsatellite allele sizes, and amplified fragment length polymorphism
(AFLP). The DNA fingerprinting data generated by microsatellite analysis were compared with
that obtained by one of the research partners (INRA, France) on protein composition of different
OSR cultivars to establish genetic variability among the cultivars tested.
For microsatellite analysis, DNA was isolated either from individual seeds of the variety
‘Express’, or from de-oiled OSR meals from 34 different cultivars. Each meal comprised of
seeds collected from a number of plants of a given variety. For AFLP analysis, DNA was
extracted from lyophilised leaf samples from individual plants of 14 different cultivars. A typical
method involved grinding around 100 mg of OSR material to a powder in liquid nitrogen using a
pestle and mortar. DNA was isolated using a NucleoSpin Plant Kit (Mache Rey-Nagel). All
DNA extracts were run on a 1.5% agarose gel to check the quantity and quality of DNA.
The primers used for microsatellite analysis (Table 1) were derived from several
members ofBrassicas family including B. nap us L. (Uzunova and Eke, 1999, Mitchell et al.,
1997).These primers were optimised for routine use. PCR amplification of microsatellite alleles
was carried out either by using DNA extracted from individual seeds or 100 mg of deoiled meal
using 'NucleoSpin Plant Kit' (Mache Rey-Nagel). The PCR conditions were 94ºC for 2 minutes,
followed by 40 cycles at 94ºC for 45 seconds, annealing at corresponding temperatures for 30
seconds, and extension at 72ºC for 1 minute. This was followed by final extension at 72ºC for 5
minutes and storage at 4ºC. The PCR products obtained by microsatellite method were separated
by electrophoresis and size of each product determined on an automated DNA analyser (ABI-
Prism-377).
CHAPTER-3
ISOLATION AND CHARACTERISATION OF
BIOACTIVE PROTEINS
3.1 Antifungal proteins:

A number of plant seeds, including Brassica spp., are known to contain the antifungal
proteins called thinness (Vernon et al., 1985). Attempts were made to isolate similar proteins
from cold-pressed de-oiled OSR meal (variety Express) using a method described by Vernon et
al. (1985). The method involved homogenising the meal in 0.1M phosphate buffer, fractionating
solubilised proteins by salt precipitation, purifying by ion-exchange Chromatography, and
desalting by gel filtration.
For this purpose, 300 g of rapeseed were initially ground using a ceramic mortar and
pestle on ice, and further homogenised in 30 g batches in 100 ml 0.1M phosphate buffer (pH 7.0)
using an Ultra Terex tissue disperser. The homogenate was frozen overnight and re-extracted in
50ml 0.1M phosphate buffer. The resulting extracts were then pooled, centrifuged at 1000g for
10 minutes and the supernatant removed. Ammonium sulphate was added at a rate of 20g per
100ml of the supernatant, left to dissolve for 10 minutes with occasional stirring and centrifuged
at 20,000g to sediment proteins (Fraction 1). The process was repeated twice with additions of
10g of ammonium sulphate per 100ml of the supernatant (Fractions 2 and 3).
The latter fractions were each suspended in 300ml 0.05M phosphate buffer (pH 7.2) with
Stirring, and were dialysed overnight in 5mM phosphate buffer (pH 7.2). A gel filtration column
was packed with 50g of pre-swollen carboxymethyl cellulose (CM52), suspended in 96 ml 0.5
phosphate buffer, using a flow rate of at least 3.7 ml/ minute. The column was equilibrated with
0.05M phosphate buffer, and 300 ml of fraction-3 were loaded onto the column. A linear
gradient of 0.1 to 1M NaCl was used to elute proteins from the column, which were collected in
15 ml fractions. The column was re-equilibrated with 0.05M phosphate buffer, and the process
repeated for fraction-2.
The fungal species Fusarium culmorum was used to determine anti-fungal activity in the
15ml fractions as thionins have been shown to specifically inhibit their activity (Terri’set
al.,1996). Four plates of sucrose nutrient agar were inoculated with F. culmorum and grown for 7
days at 25°C. Spores were harvested from the plates in 2ml sterile distilled water. The spore
suspension was filtered through a double layer of lens tissue to remove any agar fragments.0.1
ml of the F. culmorum spore suspension was spread onto plates of potato dextrose agar (PDA)
amended with streptomycin (3.165 ml of a 1% streptomycin solution in 250 ml PDA)Three
sterile filter paper circles (6 mm in diameter) were dipped into one of the extract or control
solutions and the excess wiped off. The two replicate filter paper circles were placed onto one of
the seeded PDA plates. After four days incubation at 25°C, the inhibition zone around the filter
paper circles was measured. A zero score was assigned to fractions where F.
3.2 Proteinase Inhibitors:

A protein fraction with strong proteinase-inhibitor activity was isolated from defatted
OSR meal using a method adapted from Genève et al., (1997). For this purpose, 100g of deoiled
meal was extracted with 1 litre of water using an Ultra-turrax homogeniser. The resultant
suspension was centrifuged at 3,000g for 25 minutes and the supernatant collected. The pellet 17
was re-extracted with 1 litre of water and centrifuged as above. The two supernatants were
combined and adjusted to pH 5.0 with 4N HCl. The precipitated proteins were centrifuged as
described before and the pellet discarded. The resultant supernatant was heated at 70°C for 5
minutes and cooled to room temperature. The precipitated (heat denatured) proteins were again
removed by centrifugation. The supernatant was filtered through what man No1 filter paper and
lyophilised by freeze-drying.
For further purification, the lyophilised powder was rehydrated in 200ml of 0.05M
ammonium acetate buffer (pH 5.0) containing 5% glycerol. Approximately 3ml of this extract
were filtered through 0.45μm filter (Gelman Sciences) and 2ml injected onto Sephadex G-50
column (1.5cm x 80cm) at a flow rate of 1 ml/minute, using a UK6 injector. The equilibration
and elution buffer was 0.05M ammonium acetate buffer (pH 5.0). The UV cord was set to280
nm and 1.9 ml fractions were collected continuously until no further proteins eluted from the
column.
CHAPTER-4
DNA FINGERPRINTING TYPES AND APPLICATIONS
DNA fingerprinting is one of the greatest identification systems we have to recognize an

individual or living organism. Every living creature is genetically different in its own way,
except for identical twins, triplets etc. DNA is comparable to a serial number for living things.
Each individual contains a unique sequence that is specific to that one organism. Unlike
traditional fingerprints which can be surgically altered or self-mutilated, the DNA sequence can
not easily be changed once the material is left at a crime scene, thus increasing its effective use
in forensics, and the probability of finding an exact match. This method of identification is useful
in many applications such as forensics, paternity testing, and molecular archeology, which we
will discuss later on in this chapter. To further understand DNA fingerprinting we must first
discuss the basics of DNA.
4.1 RFLPs and VNTRs:

There are three types of DNA fingerprints: RFLPs, VNTRs, and STRs. Restriction
fragment length polymorphisms, or RFLPs as they are commonly known, were the first type of
DNA fingerprinting which came onto the scene in the mid1980’s. RFLP’s focus on the size
differences of certain genetic locations .The first step in creating an RFLP fingerprint is
obtaining and isolating the DNA. DNA can be obtained from almost any of the cells or tissues in
the human body. You do not need a large amount of tissue or blood to provide enough DNA for
analysis.
The DNA is then extracted from the blood or tissue sample, and from here we carry out
our second step in the process which is the cutting, sizing, and sorting of the DNA sample. DNA
is cut using restriction enzymes, which cut the DNA stand at specific places. Restriction
enzymes are usually isolated from bacteria that use them to degrade foreign DNA like viral
DNA.
4.2 STRs and PCR:

STRs are currently the most popular type of DNA fingerprint, since the whole PCR process takes
only a few hours, compared to RFLP/VNTR probe hybridization and film exposure which can
take several days. STRs can use much smaller samples of DNA than RFLPs/VNTRs, and can
even use partially degraded DNA to create a fingerprint. Thus, the integrity and quality of the
DNA sample is not as great a factor with STRs than with the traditional methods of DNA
fingerprinting (Introduction to STRs, 2005). The current standard forensic protocol analyses 13
core STR loci which have been carefully chosen for their uniqueness. The only disadvantage of
the STR approach is it is sensitive to contaminating DNA, so usually the STR approach is used
first, followed by a VNTR analysis if contamination is suspected, and enough DNA is available.
CHAPTER-5
NEW TECHNIQUE FOR DNA FINGERPRINTING
AFLP technique is based on the amplification of subsets of genomic restriction fragments
using PCR. DNA is cut with restriction enzymes, and double-stranded (ds) adapters are ligated to
the ends of the DNA-fragments to generate template DNA for amplification. The sequence of the
adapters and the adjacent restriction site serve as primer binding sites for subsequent
amplification of the restriction fragments. Selective nucleotides are included at the 3' ends of the
PCR primers, which therefore can only prime DNA synthesis from a subset of the restriction
sites. Only restriction fragments in which the nucleotides flanking the restriction site match the
selective nucleotides will be amplified. The restriction fragments for amplification are generated
by two restriction enzymes, a rare cutter and a frequent cutter. The AFLPprocedure results in
predominant amplification of those restriction fragments, which have a rare cutter sequence on
one end and a frequent cutter sequence on the other end .
(i) The frequent cutter will generate small DNA fragments, which will amplify well and
are in the optimal size range for separation on denaturing gels (sequence gels),
(ii) The number of fragments to be amplified is reduced by using the rare cutter, since
only the rare cutter/frequent cutter fragments are amplified. This limits the number of
selective nucleotides needed for selective amplification,
(iii) The use of two restriction enzymes makes it possible to label one strand of the ds
PCR products, which prevents the occurrence of 'doublets' on the gels due to unequal
mobility of the two strands of the amplified fragments,
(iv) Using two different restriction enzymes gives the greatest flexibility in 'tuning' the
number of fragments to be amplified
(v) Large numbers of different fingerprints can be generated by the various combinations
of a low number of primers.
CHAPTER-6
DNA FORENSICS
Forensic science is the art of piecing together a crime scene in order to determine how the
crime was committed and who was responsible. DNA evidence is one of the most prominent
pieces of evidence that is used in the United States judicial system today. Just because
techniques exist that allow DNA to be analyzed at a crime scene does not necessarily mean that
evidence was collected correctly to avoid contamination, or was stored correctly to prevent DNA
degradation. As we will learn in this when we discuss landmark DNA court cases, many times
DNA evidence has been prevented from use in a particular trial due to improper handling. The
purpose of this chapter is to discuss some of the current knowledge about proper DNA handling.
DNA evidence can be collected by various means from almost any biological sample that was
left at the scene of the crime. In the past when someone committed a crime such as a sexual
assault, unless there were witnesses there was no real way of proving that a specific person was
guilty. Normal blood types are not that exclusive. Now with DNA forensics, a level of certainty
can be established that is recognized as valid evidence in a criminal case, either for the
prosecution or the defense. There have been numerous instances where men were charged with
rape in the past and had DNA analyzed from the crime scene only to find out that they were
innocent all along. shows an example of how DNA analysis can help determine who is guilty of
the crime in question. Note how the crime scene sample matches suspect 3. We will now
discuss the proper techniques to conduct a forensic investigation.
6.1 Forensic DNA and Collection Methods

DNA can be obtained from traces of biological fluids or tissues at a crime scene. The
most traditional sources of forensic DNA come from saliva, seminal fluid, blood, and hair. DNA
from saliva can be extracted from items such as cigarette butts, ski masks, envelopes, and
stamps. Seminal fluids are usually found from oral, rectal, or vaginal swabs, and also on
clothing. Blood stains and hair that are visible at a crime scene usually contain DNA that can be
analyzed at the forensic laboratory (Kramer, 2002).Collecting the DNA during a forensic
investigation may be the most crucial step in terms of the analysis results. The first step in
collecting evidence is searching for it. When examining a crime scene for evidence, a plan
should be devised to insure complete coverage of the area. For indoor crime scenes, the area will
be searched room-by-room, with each room divided into sectors. This examination technique is
called the zone method. For outdoor areas, searches are conducted by covering the area in
parallel rows, until the whole crime scene has been investigated. This technique is called the
grid method. When collecting DNA from a crime scene it is ideal to obtain the original item that
contains the evidence.
When collecting DNA evidence at a crime scene investigation, the forensic scientist
should also collect control samples to assure that the sample is pure Control samples are
specimens of any foreign substance or material that may have contaminated the DNA. The
control samples can then be analyzed separately from the other evidence to determine if any
contamination exists. When examining a crime scene for DNA one must remember to wear
protective gloves and any other necessary protection in order to prevent contamination of the
sample, and also to protect you from any possible diseases. Other kinds of collection methods
for evidence include obtaining the original items at the scene that the actual sample is on.
CHAPTER-7
STORAGE METHODS OF DNA
Biological materials are very sensitive to the conditions around them, so storage of DNA
is an important part of maintaining useful evidence. The samples acquired from the crime scene
should be kept away from high temperatures. Room temperature is acceptable, refrigeration is
desirable, and freezing is preferred when it comes to storing a sample.
DNA freezer that is used to store samples .If the samples are not stored to proper
specifications the DNA will deteriorate and will not be able to provide proper analysis. Freezers
that are “frost-free” are the most ideal when it comes to storage equipment.
7.1 DNA at an Aged Crime Scene :

At an aged crime scene, forensic scientists attempt to retrieve DNA evidence that is the
least contaminated and decomposed. For example, if a deceased individual is at an aged crime
scene and is showing signs of decomposition, then blood work would not be a practical route of
obtaining a valid DNA sample (Kramer, 2002). If hair is present on the deceased body,
thesample must be pulled from the tissue because the tissue attached to the hair root is necessary
for DNA analysis. Bones and teeth would also serve as a suitable sample of DNA evidence,
since they take very long to degrade. Another method to collect useful evidence at an aged crime
scene would be to look for property that was associated with the individual’s body such as a
hairbrush or toothbrush.Body at an aged crime scene showing some obvious decomposition.
CHAPTER-8
ADVANTAGES AND DISADVATAGES
ADVANTAGES:
 It is an easy and painless method for the subject being tested. It is less invasive then
taking a blood sample.
 It is an affordable and reliable technique.
 It can be conducted in relatively short amount of time.
 Anyone at any age can be tested with this method without any measure concerns.
 The technique has used since 1984, making it highly developed and improved.
DISADVATAGES:
 The sample of DNA can easily be ruined during the process of DNA finger printing,
causing the sample to become completely useless for testing.
 The process itself is complex and tedious, and can give result that may be hard to
interpret.
 The test needs to be run on multiple samples, a numerous amount of times for
ideal accuracy.
CHAPTER-9
CONCLUSIONS
DNA fingerprinting is the most sophisticated way to identify living organisms. DNA is a
unique piece of genetic material within biological organisms, which have Characteristicsthat are
one of a kind. DNA cannot easily be alteredonce it is left at a crime scene or deposited with a
mummy, which makes it a strong forensic tool. RFLPs and VNTRs are the traditional methods of
fingerprinting DNA, which uses a relatively large sample that uses the method of probe
hybridization to detect polymorphisms in the DNA. STRs are the most current form of DNA
fingerprinting, which is PCR based and uses a very small sample of DNA. DNA fingerprinting
has many applications that range from criminal rape cases, paternity tests, molecular
archaeology, sports memorabilia, etc. The DNA molecule is like a snowflake in that there are
no two exactly alike, but is one of the only things in common that all biological organisms are
created with.
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Sciences 13(1): 1-16.
[2] Douliez, J.P., Pato, C., Rabesona, H., Molle, D. and Marion, D. (2001) Disulfide bond
assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer
protein, LTP2, Eur. J. Biochem. 268(5): 1400-1403.
[3] Genov, N., Goshev, I., Nikolova, D., Georgieva, D.N., Filippi, B., and Svendsen, I.
(1997) A
[4] novel thermostable inhibitor of trypsin and subtilisin from the seeds of Brassica nigra:
amino acid sequence, inhibitory and spectroscopic properties and thermostability,
Biochimica et Biophysica Acta 1341: 157-164
[5] Gerbanowski A., Rabiller C.,Guéguen J. (2003) Behaviors of bovine serum albumin and
rapeseed proteins at the air/water interface after grafting aliphatic or aromatic chains, Journal
of Colloid & Interface Science 262(2): 391-399
[6] Mitchell, S.E., Kresovich, S., Jester, C.A., Hernandez, C.J. and Szewc-McFadden, A. (1997)
Application of multiplex PCR and fluorescence-based, semi-automated allele sizing
technology for genotyping plant genetic resources, Crop Science 37: 617-624
[7] Neumann, G.M., Condron, R. and Polya, G.M. (1996) Purification and mass spectrometry
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DNA FINGERPRINTING Seminar Report

2.1 DNA fingerprinting:

3.1 Antifungal proteins:

3.2 Proteinase Inhibitors:

DNA fingerprinting is one of the greatest identification systems we have to recognize an

4.1 RFLPs and VNTRs:

4.2 STRs and PCR:

6.1 Forensic DNA and Collection Methods

7.1 DNA at an Aged Crime Scene :

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