DNA FINGERPRINTING By:- Parth Shah PrinalKhimasia Shrey Shah VikashMakadya M. PHARM ( Pharmacology) NMIMS UNIVERSITY, MUMBAI.
How do you figure out that someone’s DNA is more similar to another’s? The primary method of assessing similarities is by use of DNA fingerprinting or DNA restriction analysis. This process makes use of special proteins called restriction enzymes and sections of the chromosome called tandem repeats
Definition Technology using tandem repeats of individuals to identify individuals is known as DNA fingerprinting
a person’s genetic traits, genes, are used to make specific strings of DNA letters that are cut into patterns of shorter strings separated by length these banding patterns can identify a unique human being!
Basis of genetic fingerprinting: Most of our DNA is identical to each other, however, there are inherited regions of our DNA that can vary from person to person (such variations are termed as polymorphisms). One such class of polymorphisms is known as tandem repeats, which vary within the individual of the species. This forms the basis of genetic fingerprinting.
Tandem repeats In a Eukaryotic genome, Tandem repeats are an array of consecutive repeats, known as satellites. They are of three types based on migration when centrifuged in CsCl density gradient. Satellites. Minisatellites. Microsatellites.
Satellite DNA Illustration of satellite bands. By using buoyant density gradient centrifugation, DNA fragments with significantly different base compositions may be separated, and then monitored by the absorption spectra of ultraviolet light. The main band represents the bulk DNA, and the "satellite" bands originate from tandem repeats.
SATELLITES The size of a satellite DNA ranges from 100 kb to over 1 Mb. Other satellites have a shorter repeat unit. Most satellites in humans or in other organisms are located at the centromere.
Minisatellites The size of a minisatellite ranges from 1 kb to 20 kb. One type of minisatellites is called variable number of tandem repeats (VNTR). Its repeat unit ranges from 9 bp - 80 bp. They are located in non-coding regions. The number of repeats for a given minisatellite differs between individuals. This feature is the basis of DNA fingerprinting.
Microsatellites Microsatellites are also known as short tandem repeats (STR), because a repeat unit consists of only 1-6 bp and the whole repetitive region spans less than 150 bp. Similar to minisatellites, the number of repeats for a given microsatellite may differ between individuals. Therefore, microsatellites can also be used for DNA fingerprinting.
Variable Number of Tandem Repeats (VNTR): repeats of 9 to 80 base pairs (bp), total length is 500 to 23,000 bp, very specific due to length and repeats, testing is expensive and time-consuming, degrade in older DNA samples due to random breaking of DNA strands Amplified Fragment Length Polymorphisms (AmpFLP): repeats of 8 to 16 bp, total length 100 to 1300 bp, shorter and less susceptible to degradation, first loci to be used in forensic analysis
Short Tandem Repeats: repeats of 2 to 7 bases, total length 100 to 400 bp, shorter yet thereby less susceptible to breakage, these loci are the current standard in forensic laboratory analysis, ideal size for PCR amplification Single Nucleotide Polymorphisms (SNP): a single base change as a result of mutation, not commonly useful to forensic investigators, can be potentially used to distinguish identical twins
Restriction Fragment Length Polymorphisms (RFLP) The term Restriction Fragment Length Polymorphism, or RFLP refers to a difference between two or more samples of homologous DNA molecules arising from differing locations of restriction sites, and to a related laboratory technique by which these segments can be distinguished. .
Commonly pronounced “rif-lip”.
Its analysis was the first DNA profiling technique cheap enough to see widespread application.
It is an important tool in genome mapping.
Localization of genes for genetic disorders.
Determination of risk for disease, and paternity testing
A restriction enzyme cuts the DNA molecules at every occurrence of a particular sequence, called restriction site.
For example, HindII enzyme cuts at GTGCAC or GTTAAC.
If we apply a restriction enzyme on DNA, it is cut at every occurrence of the restriction site into a million restriction fragments each a few thousands nucleotides long.
Any mutation of a single nucleotide may destroy or create the site(CTGCAC or CTTAAC for HindII) and alter the length of the corresponding fragment.
The term polymorphism refers to the slight differences between individuals, in base pair sequences of common genes.
RFLP ANALYSIS TECHNIQUE:
RFLP analysis is the detection of the change in the length of the restriction fragments.
The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digestion.
The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis.
Then transferred to a membrane via the Southern blot procedure
Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe.
Each fragment length is considered an allele, and can be used in genetic analysis.
Stages of DNA Profiling Stage 1:
Cells are broken down
to release DNA.
If only a small amount of DNA is available it can be amplified using the polymerase chain reaction (PCR).
Stages of DNA Profiling Step 2: The DNA is cut into fragments using restriction enzymes. Each restriction enzyme cuts DNA at a specific base sequence.
Stages of DNA Profiling The sections of DNA that are cut out are called restriction fragments. This yields thousands of restriction fragments of all different sizes because the base sequences being cut may be far apart (long fragment) or close together (short fragment).
Stages of DNA Profiling Stage 3:
Fragments are separated on the basis of size using a process called gel electrophoresis.
DNA fragments are injected into wells and an electric current is applied along the gel.
Stages of DNA Profiling
A radioactive material is added which combines with the DNA fragments to produce a fluorescent image.
A photographic copy of the DNA bands is obtained.
Stages of DNA Profiling Stage 4: The pattern of fragment distribution is then analysed.
Biological materials used for DNA profiling
Body tissue cells
DNA samples have been obtained from vaginal cells transferred to the outside of a condom during sexual intercourse.
APPLICATIONS 1.Paternity and Maternity person inherits his or her VNTRs from his or her parents . Parent-child VNTR pattern analysis has been used to solve standard father-identification cases 2.Personal Identification The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals.
3. 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 FBI and police labs around the U.S. have begun to use DNA fingerprints to link suspects to biological evidence – blood or semen stains, hair, or items of clothing
4.Diagnosis of Inherited Disorders diagnose inherited disorders in both prenatal and newborn babies These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others. 5.Developing Cures for Inherited Disorders By studying the DNA fingerprints of relatives who have a history of some particular disorder identify DNA patterns associated with the disease
Advantages of DNA Fingerprinting 1.Unsurpassed discriminatory potential : Complete blood group testing allows discrimiation of one person in several thousand and HLA typing one in several million DNA typing can routinely provide exclusion probabilities on the order of one in billions
DNA can be amplified
smaller sample sizes are adequate
allows rather small samples to be split and submitted for testing to more than one laboratory
3.Application to any body tissue DNA testing can be conducted with any sample having nucleated cells For example hairs, semen, urine and saliva
4.DNA is stable in comparison to proteins
resistant to degradation by common environmental insults
DNA is also long-lived in comparison to protein
Disadvantages of DNA Fingerprinting Problems with determining probability
DNA fingerprinting is not 100% assured
VNTR are results of genetic inheritance
not distributed evenly across all populations
cannot have a stable probability of occurrence.
Due to allele frequencies in different population or ethnics groups, the probability of match can range from 1 in 20 to 1 in 2 billion.
Occurrence of certain VNTR pattern depends on an individual’s genetic background.
Big problem in determining the VNTR patterns of heterogeneous genetic composition of interracial individuals
For example, the frequency of a specific allele may be 4% in Asians instead of 1% as it is in Northern Europeans.