Methods of DNA analysis

Southern Blotting: Gel Transfer

Gel-transfer hybridization—or Southern blotting—is used to detect specific DNA fragments. (A) The mixture of double-stranded DNA fragments generated by restriction nuclease treatment of DNA is separated according to length by electrophoresis

A sheet of either nitrocellulose paper or nylon paper is laid over the gel, and the separated DNA fragments are transferred to the sheet by blotting. The gel is supported on a layer of sponge in a bath of alkali solution, and the buffer is sucked through the gel and the nitrocellulose paper by paper towels stacked on top of the nitrocellulose.

As the buffer is sucked through, it denatures the DNA and transfers the single-stranded fragments from the gel to the surface of the nitrocellulose sheet, where they adhere firmly.

This transfer is necessary to keep the DNA firmly in place while the hybridization procedure (D) is carried out. (C) The nitrocellulose sheet is carefully peeled off the gel. (D)

The sheet containing the bound singlestranded DNA fragments is placed in a sealed plastic bag together with buffer containing a radioactively labeled DNA probe specific for the required DNA sequence

The nitrocellulose sheet is carefully peeled off the gel. (D) The sheet containing the bound single-stranded DNA fragments is placed in a sealed plastic bag together with buffer containing a radioactively labeled DNA probe specific for the required DNA sequence.

The sheet is exposed for a prolonged period to the probe under conditions favoring hybridization. (E) The sheet is removed from the bag and washed thoroughly, so that only probe molecules that have hybridized to the DNA on the paper remain attached.

After autoradiography, the DNA that has hybridized to the labeled probe will show up as bands on the autoradiograph. An adaptation of this technique to detect specific sequences in RNA is called Northern blotting.

In this case mRNA molecules are electrophoresed through the gel and the probe is usually a single-stranded DNA molecule

DNA fingerprinting

Like the fingerprints that came into use by detectives and police labs during the 1930s, each person has a unique DNA fingerprint. Unlike a conventional fingerprint that occurs only on the fingertips and can be altered by surgery, a DNA fingerprint is the same for every cell, tissue, and organ of a person. It cannot be altered by any known treatment. Consequently, DNA fingerprinting is rapidly becoming the primary method for identifying and distinguishing among individual human beings.

DNA fingerprinting is a laboratory procedure that requires six steps

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Isolation of DNA. DNA must be recovered from the cells or tissues of the body. Only a small amount of tissue, like blood, hair, or skin, is needed. For example, the amount of DNA found at the root of one hair is usually sufficient.

Cutting, sizing, and sorting. Special enzymes called restriction enzymes are used to cut the DNA at specific places. For example, an enzyme called EcoR1, found in bacteria, will cut DNA only when the sequence GAATTC occurs.

The DNA pieces are sorted according to size by a sieving technique called electrophoresis. The DNA pieces are passed through a gel made from seaweed agarose (a jelly-like product made from seaweed). This technique is the DNA equivalent of screening sand through progressively finer mesh screens to determine particle sizes

3) Transfer of DNA to nylon. The distribution of DNA pieces is transferred to a nylon sheet by placing the sheet on the gel and soaking them overnight.

4-5) Probing. Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint. Each probe typically sticks in only one or two specific places on the nylon sheet.

6) DNA fingerprint. The final DNA fingerprint is built by using several probes (510 or more) simultaneously. It resembles the bar codes used by grocery store scanners.

Uses of DNA Fingerprints

DNA fingerprints are useful in several areas of society. They are used by professionals in human health and the justice system.

Diagnosis of inherited disorders

DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others.

Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. In some programs, genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child. In other programs, prospective parents use DNA fingerprint information in their decisions concerning affected pregnancies

Developing cures for inherited disorders

Research programs to locate inherited disorders on the chromosomes depend on the information contained in DNA fingerprints. By studying the DNA fingerprints of relatives who have a history of some particular disorder, or by comparing large groups of people with and without the disorder, it is possible to identify DNA patterns associated with the disease in question. This work is a necessary first step in designing an eventual genetic cure for these disorders.

Forensic or criminal

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-found at the scene of a crime. Since 1987, more than 150 cases have been decided with the assistance of DNA fingerprint evidence.

Another important use of DNA fingerprints in the court system is to establish paternity in custody and child support litigation. In these applications, DNA fingerprints bring an unprecedented, nearly perfect accuracy to the determination

Personal identification

Because every organ or tissue of an individual contains the same DNA fingerprint, the U.S. armed services have just begun a program to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action. The DNA method will be far superior to the dogtags, dental records, and blood typing strategies currently in use.

Dermatoglyphics

Dermatoglyphics (from ancient Greek derma = "skin", glyph = "carving") is the scientific study of fingerprints. The term was coined by Dr. Harold Cummins, the father of American fingerprint analysis, even though the process of fingerprint identification had already been used for several hundred years

All primates have ridged skin, and it can also be found on the paws of certain mammals and on the tails of some monkey species. In humans and animals, dermatoglyphs are present on fingers, palms, toes, and soles, and give insight into a critical period of embryogenesis, between 4 weeks and 5 months, when the architecture of the major organ systems is developing.

Triradius: point of convergence of ridges from 3 different directions. Normally, there is: 1 axial triradius: normally in t, close to the wrist. 4 subdigital triradii (a.b.c.d.).

On the pad of the distal phalanx, sometimes on thenar or hypothenar eminences, are triradii, accompanied with the following patterns:
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worl: 2 triradii. loops and equivalents (ulnar or radial orientated): 1 triradius. arches: 0 triradius.

From each palmar triradius a, b, c, d, and t, is drawn the 3 lines separating the ridges at this convergence point. The longest is the main line (-- A B C D & T), ending at a side of the palm numbered from 1 to 14

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T normally ends in 13. transversality index = A+B+C+D = 27 on the Figure

Genetic disorders

Unusual dermatoglyphic patterns often relate to genetic disorders One study of foetuses with chromosomal abnormalities showed that the dermatoglyphic patterns were delayed by more than two weeks

Trisomy 21 (Down syndrome): People with Down syndrome have mainly ulnar loops, and a significantly different angle between the triradia a, t and d (the 'adt angle').

Other differences often include a single transverse palmar crease ("Simian line") (in 50%), and patterns in the hypothenar and interdigital areas, lower ridge counts along digital midlines, especially in little fingers, which corresponds to finger shortening in those with Down's syndrome

There is less variation in dermatoglyphic patterns between people with Down syndrome than between controls, and dermatoglyphic patterns can be used to determine correlations with congenital heart defects in individuals with Down syndrome by examining the left hand digit ridge count minus the right hand digit ridge count, and the number of ridges on the fifth digit of the left hand

Turner syndrome: Predominance of whorls, although the pattern frequency depends on the particular chromosomal abnormality

47, XXY (Klinefelter's syndrome): Excess of arches on digit 1, more frequent ulnar loops on digit 2, overall fewer whorls, lower ridge counts for loops and whorls as compared with controls, and significant reduction of the total finger ridge count

Trisomy 13 (Patau syndrome): Excess of arches on fingertips and single transverse palmar creases in 60%.

Trisomy 18 (Edward's syndrome) 6 - 10 arches on fingertips and single transverse palmar creases in 30%.

Cri du chat (5p-): Excess of arches on fingertips and single transverse palmar creases in 90%.

Inborn blindness : Dermatoglyphic analysis of finger tip print patterns of blind children from Bangalore

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