A Potent Weapon in Forensic Medicine

Traditionally, one of the most accurate methods for placing an individual at the scene of a crime has been a fingerprint. With the advent of recombinant DNA technology, a more powerful tool is now available:  DNA fingerprinting (also called DNA typing or DNA profiling). The method was first described by English geneticist Alec Jeffreys in 1985. 

DNA fingerprinting is based on  sequence polymorphisms, slight sequence differences between individuals, 1 bp in every 1,000 bp, on average. Each difference from the prototype human genome sequence (the first one obtained) occurs in some fraction of the human population; every individual has some differences. Some of the sequence changes affect recognition sites for restriction enzymes, resulting in variation in the size of DNA fragments produced by digestion with a particular restriction enzyme. These variations are  restriction fragment length polymorphisms (RFLPs). Another type of sequence variation, and the one now used most commonly in DNA typing, involves short tandem repeats (STRs).

Figure

The detection of RFLPs relies on a specialized hybridization procedure called Southern blotting (Fig). DNA fragments from digestion of genomic DNA by restriction endonucleases are separated by size electrophoretically, denatured by soaking the agarose gel in alkali, and then blotted onto a nylon membrane to reproduce the distribution of fragments in the gel. The membrane is immersed in a solution containing a radioactively labeled DNA probe. A probe for a sequence that is repeated several times in the human genome generally identifies a few of the thousands of DNA fragments generated when the human genome is digested with a restriction endonuclease. Auto-radiography reveals the fragments to which the probe hybridizes, as in Figure. The method is very accurate and was first used in court cases in the late 1980s. However, it requires a large sample of undegraded DNA(25 ng). That amount of DNA is often not available at a crime scene or disaster site.

The requirement for more-sensitive DNA typing methods led to a focus on the polymerase chain reaction (PCR), and on STRs. An STR locus is a short DNA sequence, repeated many times in tandem at a particular location in a chromosome; most commonly, the repeated sequences are 4 bp long. The STR loci that are most useful for DNA typing are quite short, from 4 to 50 repeats long (16 to 200 total base pairs for tetra-nucleotide repeats), and have multiple length variants in the human population. More than 20,000 tetra-nucleotide STR loci have been characterized in the human genome. More than a million STRs of all types may be present in the human genome, accounting for about 3% of all human DNA.

The polymerase chain reaction is readily applied to STR analysis, and the focus of forensic scientists changed from RFLPs to STRs as the promise of increased sensitivity became apparent in the early 1990s. The DNA sequences flanking STRs are unique to each type of STR and identical (except for very rare mutations) in all humans. PCR primers are targeted to this flanking DNA, and designed to amplify the DNA across the STR. The length of the PCR product then reflects the length of the STR in that sample. Since each human inherits one chromosome from each parent, the STR lengths on the two chromosomes are often different, generating two signals from one individual. If multiple STR loci are analyzed, a profile can be generated that is essentially unique to a particular individual. PCR amplification allows investigators to obtain DNA fingerprints from less than 1 ng of partially degraded DNA, an amount that can be obtained from a single hair follicle, a drop of blood, a small semen sample on a bed sheet, or samples that might be months or even many years old.

Successful forensic use of STR analysis required standardization. The first forensic STR standard was established in the United Kingdom in 1995. The U.S. standard, called the  COmbined DNA Index System (CODIS), was established in 1998. The CODIS system is based on 13 well-studied STR loci (Table), which must be present in any DNA typing experiment carried out in the United States. The amelogenin gene is also used as a marker. This gene, present on the human sex chromosomes, has slightly different flanking DNA on the X and Y chromosomes. PCR amplification across the amelogenin gene thus generates different-size products that can reveal the sex of the DNA donor.

Table

 Authored and Published by;

Raj Abhisek Panda

Source:(1) Adapted from Butler, J.M. (2005) Forensic DNA Typing, 2nd edn, Academic Press, San Diego, p. 96.

(2) LEHNINGER PRINCIPLES OF BIOCHEMISTRY(FIFTH EDITION),Author-David L. Nelson, Michael M. Cox, Production-W. H. Freeman and Company, 41 Madison Avenue, New York, NY 10010
*Repeat lengths observed in the human population. Partial or imperfect repeats can be included in some alleles.

*Number of different alleles observed to date in the human population. Careful analysis of a locus in many individuals is a prerequisite to its use in forensic DNA typing.

Plasmids as vectors

Plasmids are defined as autonomous elements, whose genomes exist in the cell as extrachromosomal units. They are self replicating circular (only rarely linear) duplex DNA molecules, which are maintained in a characteristics number of copies in a bacterial cell, yeast cell or even in organelles found in eukaryotic cells. These plasmids can be single copy plasmids that are maintained as one plasmid DNA per cell or multicopy plasmids, which are maintained as 10-20 genomes per cell. There are also plasmids, which are under relaxed replication control, thus permitting their accumulation in very large numbers (up to 1000 copies per cell). These are the plasmids which are used as cloning vectors, due to their increased yield potential.

Circular plasmid DNA which is used as a vector, can be cleaved at one site with the help of a restriction enzyme to give a linear DNA molecule. A foreign DNA segment can now be inserted, by joining the ends of broken circular DNA to the two ends of foreign DNA, thus regenerating a bigger circular DNA molecule that can now be separated by gel electrophoresis on the basis of its size. Selection of chimeric DNA is also facilitated by the resistance genes, which the plasmid may carry against one or more antibiotics. If a plasmid has two such genes conferring resistance against two antibodies and if the foreign DNA insertion site lies within one of these two genes, then the chimeric vector loses resistance against one antibiotic. In such a situation, the parent vector in bacterial cells can be selected by resistance against two antibodies and the chimeric DNA can be selected by retention of resistance against only one of the two antibiotics. 

Authored and Published by;

Raj Abhisek Panda

Cloning Vectors for Recombinant DNA

One of the important uses of recombinant DNA technology is the cloning of (i) random DNA or cDNA segments, often used as probes or (ii) specific genes, which may be either isolated from the genome or synthesized organochemically or in the form of cDNA from mRNA. This cloning of DNA is possible only with the help of another DNA molecule, which is capable of replicating in a host. This other DNA molecule is often used in the form of a vector, which could be a plasmid, a bacteriophage, a derived cosmid or phagemid, a transposon or even a virus. Techniques should also be available, which will allow selection of chimeric genomes obtained after insertion of foreign DNA from a mixture of chimeric and the original vector. Another critical desired feature of any cloning vector is that it should possess a site at which foreign DNA can be inserted without disrupting any essential function. Therefore, in each case an enzyme will also have to be selected which will cause a single break. Sometimes vectors ate modified by inserting a DNA segment to create unique site(s) for one or more enzymes to facilitate its use in gene cloning. This DNA with restriction sites for several enzymes is sometimes called a polylinker.

Authored and published by;

Raj Abhisek Panda 

Recombinant DNA and Gene Cloning

Cloning and Expression Vectors

In recent years, techniques for manipulating prokaryotic as well as eukaryotic DNA have witnessed a remarkable development. This has allowed breakage of a DNA molecule at two desired place to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position. The product thus obtained is called Recombinant DNA and the technique often called genetic engineering. Using, this technique we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies, all identical. This become possible because vectors like plasmid and phages reproduce in a host (e.g. E.coli) in their usual manner even after insertion of foreign DNA, so that the inserted DNA will also replicate faithfully with the parent DNA. This technique is called gene cloning. With this technique, genes can be isolated, cloned and characterized, so that the technique has led to significant progress in all areas of molecular biology.

A variety of vectors have been developed which not only allow multiplication, but may also be manipulated in such a way that the inserted gene may express in the host. Due to the importance of a variety of these cloning and expression vectors in genetic engineering experiments, they will discussed in some detail in next posts.  The techniques used for inserting foreign DNA in these vectors and the development of chimeric DNA molecules (for developing molecular probes, gene libraries, etc.) will be discussed in subsequent posts.

Authored and published by

Raj Abhisek Panda

 References;

Elements of BIOTECHNOLOGY, P.K. GUPTA. 2002,Title code no.-BC-22,PP no-14