Locating Genes With Recombinant DNA Techniques


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This is a restriction map of pBR322 Plasmid. Image by Ayacop, Yikrazuul

The Human Genome Project, completed in 2002, succeeded in sequencing the 3 billion DNA base pairs and approximately 20,500 genes (sequences of base pairs) in human chromosomes. This has opened up endless possibilities in areas as diverse as medicine, evolutionary research, agriculture and forensic science, but how does it work?

Over the last few decades, several recombinant DNA techniques have facilitated the location of positions of the actual genes on each chromosome of various organisms. These techniques include restriction mapping, hybridization using radioactive probes, the Maxam -Gilbert method and the Sanger technique for DNA sequencing.

All techniques rely to some degree upon recombinant DNA technology – namely, the use of the enzymes DNA polymerase and DNA ligase, along with specific ‘restriction’ enzymes to cut and re-join fragments of DNA. We then separate these fragments  using electrophoresis, and locate them using radioactive or fluorescent probes. More advanced separation and detection methods have also recently been developed.

DNA Technology: Restriction Mapping

In this process, which is used mainly for short genomes such as bacteria, we cut known lengths of bacterial plasmids (loops of bacterial DNA) with different restriction enzymes. Because each restriction enzyme cuts at a specific base sequence, we can then determine the orientation of these restriction sites. Combining this with DNA hybridization techniques, using labelled probes of a known sequence, allows us to deduce the base sequence of the whole strand.

Although researchers still use these combined methods in situations where more advanced DNA sequencing techniques are unnecessary (usually for short genomes), we mainly use restriction mapping to locate the position of DNA inserts in recombinant bacterial plasmids.

DNA Hybridisation Techniques

In DNA hybridisation, scientists use fluorescent or radioactive markers to label segments of DNA with known sequences. They then hybridize these ‘probes’ with single strands of segments of the unknown DNA. Successful hybridisation occurs when bases from the probe match with their complementary bases on the DNA strand. Using this information, and knowledge of the fragment’s position on a DNA restriction map, researchers can then locate a particular gene on a chromosome.

Alternatively, the FSH (fluorescence in situ hybridisation) technique can introduce these probes directly to chromosomes on a microscope slide. After washing away unmatched probes, we can detect the areas where hybridisation has occurred by using a fluorescence microscope.

Gene Linkage Maps

This image illustrates meotic recombination for Human Chromosome 1. Image by EMW

Genetic linkage maps rely on the premise that the further apart two genes are on a chromosome, the more likely the chromsomes are to ‘cross over’ in the region between them during meiosis. Alfred Sturtevant recognized that this distance could be measured as a function of the number of recombinant offspring produced for a particular gene.

Although this method is feasible in bacterial and plant studies, in human genome studies. it is obviously impractical to control matings and to wait for the outcomes over several generations. Researchers have therefore only applied linkage maps to the study of hereditary diseases in humans.

The Maxam–Gilbert Method

In this process, researchers chemically cleave sections of the DNA template strand, one nucleotide before a specific base. They then separate these segments on an electrophoresis gel according to base length.

The position of each nucleotide will therefore be one base length longer than the section from which it was cut. Although outdated, this process paved the way for the more practical Sanger technique, used in the Human Genome Project.

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