Is it Possible that DNA Genetic Material itself
to fool the Definite Determination? Part VII
Real Fathers may escape responsibility of fathering; Lawyers shall stand on their toes on floor to question the protocols used by laboratories, to determine a real father.
First
of all I render my apologies, for some typing mistakes in the Part VI, which
rendered unable to understand, what I have described in the beginning of the
procedure for Polymerase Chain Reaction (PCR) fortunately
though the typing mistakes has not alter the meaning or the essence of
techniques explained; now it has been corrected and here uploaded a you-tube
link for further acquainting to PCR.
In the Part V, I was
discussing the earliest method of DNA typing, the RFLP process and the main
reasons for it had been supplanted by PCR and the emergence of a technique,
which was termed as Genetic Finger Printing, rather inadvertently by Prof. Alec
Jeffrey Sir, who introduced the using of Introns of DNA and as well adding a
barcode technique for interpreting of DNA analysis. Within months of Prof. Alec
Jeffreys’ scientific papers in 1985, it was brought to the floor of the court
through a lawyer to solve a longstanding immigration dispute of a Ghanaian
family of UK citizens. The family's youngest son had recently returned from a
trip to Ghana, but the Home Office detained him, alleging that he held a forged
passport, and claimed that the boy is not born to that parents. Jeffreys compared the DNA from the boy, the woman who claimed
to be his mother, and her other children through a multi-locus
restriction fragment length polymorphism (RFLP) of variable loci and it
proved that the
boy was the woman’s son and that all of
her children shared the same father and the immigration was allowed within UK.
Jeffreys went on to settle an unresolved rape murder case within Leicester
Canterbury among the two suspects one was convicted on both accounts, other one
was exonerated absolutely. Elsewhere in Italy another rape and murder which had
not been resolved for a grueling 14 years, brought to book by RFLP –genetic
fingerprinting, through a dried up chewing-gum sample from the site of crime
after 14 long years after the crime. In USA one who was convicted for Rape and
sentenced 245 years RI, was exonerated from all charges after spending his
7 years in jail and the list goes on and on. Still European Law emphasizes that it is
mandatory to obtain a court order before undertaking or storing genetic finger
printing of an individual by any method, quite an absurd mandate since
introns can never reveal anybody’s personal characterizations.
Though RFLP method is laborious, time consuming
and require larger DNA, and hence exposed to human errors, it does not require a known DNA sequence as in the case of
PCR, which requires to be added with Primers containing sequences
complementary to the target region along with a DNA
polymerase, in order to elongate the chain, and thus
RFLP only sparingly used only when reference sample of unknown originates.
The Story of Emergence Short
Tandem Repeat (STR) Method.
The
RFLP technique became the first scientifically accepted forensic DNA analysis
method, but it had a few drawbacks: the single locus probe strategy still
required relatively large amounts of DNA, and that DNA had to be recovered from
a crime scene in a high quality form. DNA degrades when exposed to the
environment, breaking into fragments that may be too small for RFLP analysis.
Researchers overcame
these limitations in two stages. In the first, scientists applied the new
polymerase chain reaction (PCR) technique to forensic analysis since the PCR
method can duplicate, or amplify, a billion copies of a target nucleotide
sequence, even a target sequence buried in a mixture of millions of other
sequences and PCR duplication can be performed with a Nano-gram of DNA in
contrast to 10Micrograms- for RFLP a 10 x 10³ fold reduction of required
quantity.
But still there was a hitch. PCR could duplicate only DNA
segments much shorter than numerous mini-satellite targets. Therefore
incorporation of PCR amplification into forensic DNA analysis required a switch
to a smaller targets that offered the same type of tandem repeat variation.
Here where the stage two comes in: the use of microsatellites. While
minisatellites have sequences of six to 100 nucleotides bp, that repeat two to
several hundred times, microsatellites have repeat unit lengths that typically
peak at seven nucleotides, repeated five to 100 times. (The entire probable
target intronic codon sequences are referred to as minisatellites and
microsatellites)
In 1991, Baylor College
of Medicine's Thomas Caskey suggested using a type of microsatellite called
short tandem repeats (STRs).
We all know that the human genome is full of repeated DNA
sequences. These repeated sequences come in various sizes. DNA regions with
short repeat units of introns (usually 2-6 bp in length) are called Short
Tandem Repeats (STR). STRs are found surrounding the chromosomal
centromere (the structural center of the chromosomes)STRs, are repeated in tandem fashion
around five to a few dozen times and are classified according to the length of the core
repeat units, the number of adjoining repeat units, and/or the overall length
of the repeat region. Each
repetition number is called an allele. Thus, a 10-fold repetition of the core unit at locus X is called
‘allele 10 of locus X’. The combination of STR alleles at several loci is
unique for an individual, even if some or many alleles can be shared with
others.
Peter Gill, then a
researcher at Britain Forensic Science Service, devised the PCR-STR method,
which enabled the simultaneous analysis of multiple STRs, a tactic called multiplexing.
With this technique at hand, investigators could analyze DNA recovered
from minute and even partially degraded samples. Forensic DNA analysis
no longer depended upon the availability of tissues and fluids, such as blood
and semen. Now, DNA analysis could be performed with PCR-amplified
biological traces acquired from dental molds, cigarette butts, eating utensils,
chewing gum, postage stamps, ski masks, licked envelopes, toothbrushes, razor
shavings, band aids, and real palm fingerprints.
If full proof of a match between the genetic fingerprint from a
biological sample from a crime scene and the DNA profile of an offender is
needed, sometimes the combination of STR alleles from up to 12–15 loci might be
needed (Figure3) This, however, will lead to the perfectly safe statement that ‘no
other person on this continent/on earth shares the same allele composition’.
VNTR- Variable Number of
Tandem Repeat
Flanking
the Short Tandem Repeats (STRs) in the chromosome are VNTR Variable Number
of Tandem Repeats, as the name
implies sequences of tandem repeat varies in individuals as well as within
tissues of individuals. It can be added or removed by replication or recombination
errors giving rise to varying number alleles of repeats.
VNTR blocks can be extracted with restriction enzymes and analyzed by RFLP, or amplified by the polymerase chain reaction (PCR) technique and their size determined by gel electrophoresis.
VNTRs were an important source of RFLP genetic markers used in linkage analysis (mapping) of diploid genomes. Now that many genomes have been sequenced, VNTRs have become essential to forensic crime investigations, via DNA fingerprinting and the CODIS* database.
*CODIS = Combined DNA Index System (CODIS) is a FBI's program of support for criminal justice DNA
databases as well as the software used to run these databases, it is just an
computing technology extension for DNA analysis applied only in USA and Canada.
When removed from surrounding DNA by the PCR or RFLP methods,
and their size determined by gel electrophoresis or Southern blotting, they produce a
pattern of bands unique to each individual. When tested with a group of
independent VNTR markers, the likelihood of two unrelated individuals' having
the same allelic pattern is extremely low. VNTR analysis is also being used
to study genetic diversity and breeding patterns in populations of wild or
domesticated animals. As such, VNTRs can be used to distinguish strains of
bacterial pathogens. In this microbial forensics context, such assays are
usually called Multiple Loci VNTR Analysis or MLVA.
In
analyzing VNTR data, two basic genetic principles can be used:
Identity
Matching- both VNTR
alleles from a specific location must match. If two samples are from the same
individual, they must show the same allele pattern.
Inheritance
Matching- the VNTR
alleles must follow the rules of inheritance. In matching an individual with
his parents or children, a person must have an allele that matches one from
each parent. If the relationship is more distant, such as a grandparent or
sibling, then matches must be consistent with the degree of relatedness.
Next Generation works on Exons
Before I wind up, it is quite appropriate to stress that DNA analysis, despite it is being expanded day by day through continuous research universally, basically there are only two analytical procedure for forensic studies, that are RFLP and PCR method; all the other procedures that explained are of just a difference in target sequence codons or markers of the DNA for above two procedures to obtain a results in bands, sometimes illuminated for clarity. Gene codons, noncoding introns, STR, and VNTR are all DNA markers for analysis through RFLP and STR. The researchers are very keen in bringing in another target or marker of DNA popularly known amongst Molecular Biologists, Geneticists and specially the Forensic Scientists as EXONS into that list.
What are EXONS? Exons are both the DNA
sequence within a gene and to the corresponding sequence in RNA
transcripts, that would be lost to the mature mRNA; that means gene codons just
completed expressing (a message) along with corresponding trRNA and mRNA codons
that lost the Introns through RNA splicing. If they progress to succeed targeting
the sequences of Exons, they might even progress to detect what kind of proteins
have been expressed by those genes since the crime, as well, leading into the behavioral
pattern of suspects. Long way to go and there would be an ethical barrier as
well!
Coming up will be pairing, Sexing and Mutation
for selection in the final episode
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