Analysis of the Human Genome | Genetics

In this article we will discuss about the analysis of the human genome.

Molecular analyses of medical relevance rely heavily on four strategic features:

(1) The ability to clone and characterize nucleic acid sequences,

(2) The specificity of nucleic acid hybridization,

(3) The specificity of restriction endonucleases, and

(4) The power of DNA amplification using the polymerase chain reac­tion (PGR).

Various combinations of these capabilities are used to accomplish diverse diagnostic procedures.

Molecular Cloning and DNA Sequencing:

The size, the complexity, and the variability of the human genome constitute barriers to the analysis of individual traits and genes. The feasibility for such analysis was greatly enhanced by the development of recombinant DNA technology.

This technolo­gy allows for the analysis of one DNA frag­ment at a time and has provided an “index” for the human genome and made it possible to locate and isolate individual genes, segments of genes, or nucleotide sequences from the vast DNA library.

One of the most powerful of these tech­niques, so-called cloning of DNA, makes it possible to isolate individual genes or portions of genes, to make an unlimited number of copies of such DNA fragments, to determine the nucleotide sequence of the DNA, and to transcribe and translate the genes so isolated.

The various genes and gene products can then be utilized for diverse studies of gene structure and function in normal and diseased states. Other recombinant DNA techniques, restric­tion enzyme digestion, and DNA amplifica­tion, to be described below, disease traits can be analyzed both directly and by genetic link­age to polymorphic sites.

The cloning of DNA involves isolation of DNA fragments and insertion of a sequence into the nucleic acid from another biologic source (vector) for manipulation and propaga­tion. The most widely used vectors are based on bacterial plasmids or bacteriophage such as phage λ, or M13, some variations of which can accommodate DNA fragments up to 45 kb in size.

In addition, vectors designed to function as yeast artificial chromosomes (YAC vectors) can be utilized for cloning DNA frag­ments up to hundreds of kilobases in size. Space does not allow presentation of the var­ious methods for DNA cloning, but these are well described in Watson, and in Sambrook.

For purpose of this discus­sion, it is adequate to point out that cDNA (DNA complementary to mRNA) clones have been isolated for hundreds of human genes. Most of these cDNAs have been cloned after characterization of the gene product to obtain amino acid sequence data or specific antibod­ies. Genomic DNA clones are also available for hundreds of human genes.

In addition, many hundreds of anonymous genomic DNA clones are in widespread use. These anony­mous DNA clones are generally of interest because they detect polymorphisms that map to a particular location in the human genome, as will be discussed below.

Human genomic DNA libraries have been prepared containing most or all of the human genome in fragments of 15 to 45 kb. Radioactive, biotinylated, or otherwise modi­fied copies of DNA can be prepared from any cloned fragment and can serve as a specific molecular probe.

Biotinylated probes can be detected using avidin and secondary detection methods. Cloned DNA can be sequenced using manual or semi automated methods, and the sequence of millions of base pairs of human DNA is already known, although only about 0.1 percent of the human genome was sequenced as of early 1989.

If a gene or a genomic segment has been cloned, it is rela­tively straightforward to clone the same region from individual patients and determine the sequence for any mutation. The availabil­ity of sequence data allows for the use of the polymerase chain reaction for DNA amplifi­cation.

Thus, some of the fruits of molecular cloning are the availability of probes for ana­lytical procedures, the determination of dis­ease mutations, and the availability of sequence data to allow for DNA amplifica­tion. Cloned DNA can also be used for pro­duction of proteins, for detection of sequences of infectious organisms, and for research activities.

Nucleic Acid Hybridization:

Many of the steps in recombinant DNA anal­ysis take advantage of the fact that the complementary nature of nucleic acid interaction is the result of base pairing during the synthe­sis of DNA and RNA. Linear pieces of double- stranded (native) DNA can be treated with heat or alkali to dissociate the two strands to yield single-stranded (denatured) DNA.

The denatured DNA can be incubated under con­ditions that allow for nucleic acid hybridiza­tion, i.e., the recognition of two complemen­tary strands and re-formation of double stranded molecules by base pairing. Nucleic acid hybridization is so sensitive that a single stranded DNA molecule can be hybridized specifically to a complementary strand of RNA or DNA present at about 1 part in 10,000.

Many recombinant DNA studies involve the preparation of one radioactive or biotinylated strand of nucleic acid which is then used as a “probe” in the analysis. It is possible to identify and distinguish both fully homologous sequences and partially homolo­gous sequences.

The specificity of nucleic acid hybridization, often in combination with frac­tionation or amplification procedures, allows detection of a single gene among tens of thousands or of a viral sequence in the midst of other nucleic acid sequences.

A variation on nucleic acid hybridization involves the use of allele specific oligonu­cleotides (ASO):

The DNA probe is a syn­thetic, single-stranded oligonucleotide, usually 15 to 20 bases in length. The oligonu­cleotides are synthesized to be complemen­tary to each of two or more sequences that represent polymorphisms or mutations in the genome.

The variable nucleotide is in the mid portion of the oligonucleotide. Hybridiza­tion conditions are adjusted so that the oligonucleotide detects a perfectly matched sequence but fails to hybridize if there is a sin­gle base mismatch.

Allele-specific oligonu­cleotides can be used in combination with DNA amplification, as described below. The majority of hybridization proves have been prepared in radioactive form, but the use of nonradioactive detection methods is likely to increase.

Restriction Endonucleases:

The discovery, in microorganisms of restric­tion endonucleases, commonly known as restriction enzymes, facilitated recombinant DNA manipulations. The enzymes recognize a specific oligonucleotide sequence in double- stranded DNA and cleave the DNA at this site.

Many enzymes are known, each recog­nizing a unique DNA sequence. Some; enzymes recognize sequences only four base pairs in length. For example, the enzyme Hae III cleaves the sequence 5′-GGCC-3′. By con­vention, only one strand of DNA is printed as the recognition site, but the enzymes recog­nize double-stranded DNA.

Other enzymes recognize sequences six base pairs in length. The enzyme Hind III cleaves the sequence 5′- AAGCTT-3′. Other enzymes, such as Mst II, recognize a seven-base-pair sequence but tolerate any base pair in the middle position.

The sequence specificity of restriction enzymes is a powerful tool in dissection of large genomes. When human DNA is diges­ted with a particular restriction enzyme, hun­dreds of thousands of DNA fragments are generated with remarkable reproducibility.

Such fragments can vary from a few base pairs to several thousand base pairs in length, depending on the enzyme used. Restriction enzymes that recognize a sequence only four base pairs long cleave the DNA into smaller fragments than enzymes that recognize a six- base-pair sequence.

With the use of multiple restriction enzymes to analyze a particular segment of DNA, it is possible to define a detailed map of restriction endonuclease cleavage sites for the region. Such a map can span a region of from several hundred to tens of thousands of base pairs of DNA. As described below, variations in the sequences of those cleavage sites can be analyzed as polymorphisms or mutations in the human genome.

Southern Blotting:

Many analyses of the human genome involve a specific appli­cation of DNA-DNA hybridization, the blot­ting procedure developed by E.M. Southern. For clinical analysis, Southern blotting (Fig. 19.2) begins with the isolation of genomic DNA from cells such as peripheral leukocytes or fetal cells.

The high-molecular-weight genomic DNA is digested with a restriction enzyme to yield a series of reproducible frag­ments. These DNA fragments are separated by electrophoresis in agarose gels. After elec­trophoresis, the DNA is transferred from the gel to a membrane that binds the DNA.

The membrane is treated to denature the DNA and is soaked in a solution containing a radioactive single-stranded nucleic acid probe. The probe will form a double-stranded nucle­ic acid complex at sites on the membrane where homologous DNA is present. The membrane is washed to remove unbound radioactivity, and regions on the membrane where homologous DNA sequences were bound are detected using X-ray film.

The sen­sitivity of southern blotting is achieved by splitting the DNA into small segments, frac­tionating the fragments, and applying a sensi­tive detection method to pick out specific fragments (nucleic acid hybridization).

Overall this method can detect genomic DNA frag­ments that represent a single gene or about 1 part in 1 million in the genome. The clinical power of Southern blotting resides in the abil­ity it gives to analyze a tiny portion of the pri­mary structure of human genomic DNA taken from an individual.

An analogous procedure starting with RNA for analysis has been termed northern (in contrast to southern) blotting. In this pro­cedure, the presence or absence of a particu­lar mRNA as well as its approximate size can be determined.

The term immuno-blotting or Western blotting describes a derivative pro­cedure designed to analyze protein antigens. Proteins are separated by electrophoresis and transferred to a solid membrane through a blotting procedure. The membrane is ana­lyzed by incubation with antibodies followed by a second step for enzymatic or radioactive detection of bound antibody.

Thus, southern blotting, northern blotting and immuno-blotting or western blotting each combines a frac­tionation and a detection method to provide a sensitive technique for the analysis of DNA, RNA, and protein, respectively (Table 19.1).

Polymerase Chain Reaction (PCR) For DNA Amplification:

The tech­nique of PCR for DNA implication is a power­ful method that has had a revolutionary impact on molecular diagnosis. The method was pio­neered and patented by workers at the Cetus Corporation. The technique is based on know­ing the nucleic acid sequence for a region which, for a diagnostic application, is to be analyzed repeatedly. Oligonucleotide primers are prepared that are complementary to oppo­site strands of the DNA and are separated by up to a few hundred base pairs.

The oligonu­cleotide primers are incubated with the target DNA to be amplified and with a DNA poly­merase that synthesizes a complementary strand in a 5′-to-3′ direction. Considerable specificity is provided by the requirement that primers must lead to convergent synthesis for amplification to be effective.

The reaction is subjected to a series of temperature variations including a denaturing temperature where dou­ble stranded DNA is dissociated to single- stranded DNA, an annealing temperature where oligonucleotide primers hybridize to target DNA, and a polymerization temperature for the synthetic step.

The reaction is usually carried out using heat-resistant Taq (from Thermus aquaticus) polymerase such that the polymerase remains active during the temperature cycles (usually ranging from 50° to 95°C). After a number of such cycles— typically 20 to 30 or more—hundreds of thou­sands of copies of the original target sequence are synthesized. The bulk of the product is a double-stranded DNA fragment of specific length.

The technique has been used to amplify and analyze DNA from a single human sperm which contains one duplex target DNA molecule. Molecular diagnosis with PCR depends on determining the presence or the absence of an amplified product, digesting the amplified product with a restriction enzyme, hybridizing the PCR product with allele- specific oligonucleotides and direct sequencing of the PCR product, or on other methods of analysis.

Many variations and modifications have been devised to take advantage of the PCR concept. These include synthesis of cDNA with reverse transcriptase followed by amplifi­cation of the cDNA. Single-stranded DNA can be synthesized by altering the ratio of the oligonucleotide primers.

Research applications include preparation of recombinant DNA constructs, mutagenesis of cloned DNA, detection of rare nucleotide sequences, and detection of nucleotide sequences of infectious agents. The PCR method offers extremely rapid analysis (single day), case of automation, relative economy, and extraordinary specificity.