Name : Nadhifa Salsabila

NIM : 185100500111003
Class : Biotechnology (RE)

Restriction Fragment Length Polymorphism (RFLP)

The basic principle of RFLP is separation of the desired repetitive sequences by cleaving
them out from the genome using an appropriate restriction endonuclease enzyme,
electrophoresis of the digested DNA and thereafter detection of DNA bands. Traditionally,
the RFLP test has been the method of choice for DNA typing. While RFLP testing is very
accurate for some types of specimens, such as fresh whole blood, it frequently is not suitable
for aged or environmentally stressed specimens, samples with a limited amount of DNA such
as traces of saliva recovered from cigarette butts, and bone. In present analysis the restriction
sites for ECORI & PST enzyme mixture were not located at the same positions in blood and
hair DNA samples. The RFLP technique uses VNTRs. VNTRs are regions of DNA
comprising hundreds to several thousand base pairs and are arranged as tandem repeat units.
Loci with long motifs (e.g., 8-80 bp) are referred to as minisatellites or variable number of
tandem repeats (VNTRs). The number of repeats varies greatly from person to person.
Restriction enzymes were used for cleaving VNTR fragments varying in lengths, which were
detected by the RFLP technique (Rahiman, 2014).

Application of RFLP test (Rahiman, 2014) :

 Genome mapping : helps in analysis of unique pattern in genome for organism
identification and differentiation. It also helps in determining recombination rate in
the loci between restriction sites.
 Genetic disease analysis : After identification of gene for particular genetic or
hereditary disease, that gene can be analyzed among other family members.
 To detect mutated gene.
 DNA finger printing (forensic test) : It is the basis of DNA finger printing for
paternity test, criminal identification etc.
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Polymerase Chain Reaction (PCR)

PCR is a method of making unlimited copies of DNA using a test tube, a heat source and few
simple reagents [template, primers, DNA polymerase, deoxyribonucleoside triphosphate
(dNTPs) and a buffer]. In technical terms however, PCR may be defined as a rapid procedure
for in vitro enzymatic amplification of specific DNA sequences (in pure form or complex
mixtures) using two oligonucleotide primers that hybridize to opposite strands, and flank the
region of interest in the target DNA. The PCR can generate 100 billion similar copies.
Broadly, the applications of PCR in the biological sciences may be divided into medical
application and research application (Atawodi, 2010).

Specific applications of PCR in the medical sciences include (Atawodi, 2010):

 Diagnosis of Monogenic Diseases,
  Diagnosis of Mutation Diseases,
 DNA Typing, Evolutionary Trends and Disease Susceptibility Studies
 PCR and Forensic Science
 Detection of Human Infectious Diseases
 Detection of ras Oncogenes
 PCR and DNA Vaccine Production

Most examples of applications of PCR in scientific research may be summarized as follows

(Atawodi, 2010) :
 Direct sequencing of in vitro amplified DNA
 Engineering DNA to meet specific needs
 Detection of mutation.
 Detection of gene expression
 Specific amplification of a DNA specie
 Geometric amplification of unknown DNA sequence through inverse PCR
 Analysis of DNA sequences in individual gametes
 Evolutionary analysis
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Short Tandem Repeat (STR)

STR analysis is the method most widely used in forensic science. This type of DNA analysis
works to examine individual areas in DNA. The differences for certain DNA regions in one
person versus another can allow for distinguishing between individuals. STR analysis
involves the extraction of nuclear DNA from cells in a sample. Certain regions of the DNA
that are extracted are then amplified by the PCR. After amplification, a scientist performs gel
electrophoresis to find out how many repeats of the STR sequence exist. Conventional PCR
was used for STR typing, and the PCR products, consisting of amplified STR loci
(amplicons) were electrophoresed with a DNA analysis device. About ten STR markers were
used as standards for STR characterization and analysis of size. STR polymorphisms involve
a large number of alleles that enable the analysis of multiple loci, thus enhancing the
probability of individual identification (Lim, 2015).

 STR analysis is still somewhat of a new technology in forensic science. Currently, it

is a popular method for genetic profiling and STR analysis used today involves four to
five nucleotide repeats, which allows scientists to obtain relatively precise and
accurate information (Butler, 2012)
 STR can be successfully used to monitor patients following transplant therapy. It can
allow for the prediction of problems associated with transplants, such as graft
rejection or a relapse of the disease. This proactive type of testing can significantly
aid the patient in obtaining recovery from their condition and transplant surgery
(Butler, 2012).
 BIBLIOGRAPHY

Atawodi, SE., et al. 2010. Polymerase Chain Reaction: Theory, Practice and Application: A
Review. Sahel Medical Journal. Vol. 13(2): 54-63

Butler, John M. 2012. Short Tandem Repeat (STR) Loci and Kits. Journal of Biotechniques.
Vol. 43(2): 99-110

Lim, Seri., et al. 2015. Characterization of Human Short Tandem Repeats (STRs) for
Individual Identification Using the Ion Torrent. BioChip Journal. Vol. 9(2): 164-172

Rahiman, Shaik., et al. 2014. Restriction fragment length polymorphism (RFLP) application
in DNA typing for Crime investigation. Indian Journal of Forensic Medicine &
Toxicology. Vol. 4(1): 79-82