Recombinant DNA Technology DNA Libraries

A DNA library is a collection of cloned restriction fragments of the DNA of an organism. There are two kinds of libraries: genomic libraries and complementary DNA (cDNA) libraries. Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome. In contrast, cDNA libraries contain those DNA sequences that appear as mRNA molecules, and these differ from one cell type to another. Cloned cDNA lack introns and the control regions of the genes, whereas these are present in genomic libraries.

A genomic library is the collection of fragments of dsDNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. The recombinant DNA molecules are replicated within host bacteria. The amplified DNA fragments represent the entire genome of the organism and are called a genomic library.

cDNA libraries: In certain types of tissues, there is a gene (gene of interest) that is highly expressed, and its corresponding mRNA is also present at high concentrations in the cell e.g reticulocyte mRNA is composed largely of molecules encoding the α-globin and β-globin chains of hemoglobin. This mRNA can be used as a template to make a complementary dsDNA (cDNA) molecule using the enzyme reverse transcriptase.

This mRNA is isolated from transfer RNA (tRNA) and ribosomal RNA (rRNA) by the presence of its polyA tail. The resulting cDNA is thus a double-stranded copy of mRNA. cDNA can be amplified by cloning or by the polymerase chain reaction (PCR). Later on, it can be used as a probe to locate the gene that coded for the original mRNA (or fragments of the gene) in mixtures containing many unrelated DNA fragments. As the cDNA has no intervening sequences, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria.

Probes A probe is a short, single-stranded piece of DNA, labeled with either a radioisotope, such as 32P, or with a nonradioactive probe, such as biotin. The nucleotide sequence of a probe is complementary to the DNA of interest, called the “target DNA”. Probes are used to identify which clone of a library or which band on a gel contains the target DNA.

The utility of probes depends on the phenomenon of hybridization (or annealing) in which a single-stranded sequence of a target DNA binds to a probe containing a complementary nucleotide sequence. ssDNA of the target DNA which is produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane.

The immobilized DNA strands are prevented from self-annealing, but are available for hybridization to an exogenous, single-stranded, radiolabeled DNA probe. The extent of hybridization is measured by the retention of radioactivity on the membrane. Excess probe molecules that do not hybridize are removed by washing the filter.

If the sequence of all or part of the target DNA is known, single-stranded oligonucleotide probes of 20–30 nucleotides can be synthesized that are complementary to a small region of the gene of interest. If the sequence of the gene is unknown, the amino acid sequence of the protein—that is, the gene product—may be used to construct a probe. Short, ssDNA sequences (15–30 nucleotides) are synthesized, using the genetic code as a guide. Note: Degeneracy (redundancy) represents a problem here, why? And what is the solution?

Example of clinical use of Probes An allele-specific oligonucleotide (ASO) probe can be used to detect the presence of the sickle cell mutation in the β-globin gene. What is sickle cell disease? In normal adults, hemoglobin is HbAº which consists of 2 alpha and 2 Beta chains. The sixth a.a. sequence of the ß- globine chain towards the amino terminal end this chain is glutamine. Mutations can alter the codon sequence that codes for the glutamine by another that codes for valine. The altered ß- globine chain is called as ßs- chain. The resultant Hb is (Hb S) with a low affinity to O2 and RBC will be sickle shaped.

DNA, isolated from white blood cells, is denatured into single strands DNA, isolated from white blood cells, is denatured into single strands. An oligonucleotide (i.e. ASO) is constructed that is complementary to the portion of the mutant globin gene coding for the amino-terminal sequence of the β-globin protein. DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell disease) contains a nucleotide sequence that is complementary to the probe. Thus, a double-stranded hybrid forms can be detected by electrophoresis.

In contrast, DNA obtained from normal individuals is not complementary at the sixth nucleotide triplet and, therefore, does not form a hybrid. Note: A pair of ASO probes has to be added; one for the normal allele and one for the mutant allele.

Antibodies Sometimes no amino acid sequence information is available to guide the synthesis of a probe for direct detection of the DNA of interest. In this case, a gene of interest can be identified indirectly by cloning cDNA in an expression vector that allows the cloned cDNA to be transcribed and translated to a certain protein product. A labeled antibody is used to identify which bacterial colony produces the protein and, therefore, contains the cDNA of interest. [Note: A library created using expression vectors is called an expression library.]

Southern Blotting Technique It is one of the technique used to detect mutations in DNA. It is called so because it was invented by Edward Southern. It involves the following steps: 1. DNA is first extracted from cells, e.g a patient's leukocytes. 2.DNA is cleaved into many fragments using a restriction enzyme. 3. The resulting fragments are separated on the basis of size by electrophoresis. [Note: Large fragments move more slowly than the smaller fragments].

4. The DNA fragments in the electrophoresis gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. 5. The last step a probe to identify the DNA fragments of interest. The patterns observed on Southern blot analysis depend both on the specific restriction endonuclease and on the probe used to visualize the restriction fragments.

[Note: Variants of the Southern blot have been named “Northern” (electrophoresis of mRNA followed by hybridization with a specific probe), and “Western” (electrophoresis of protein followed by detection with an antibody directed against the protein of interest].

Prenatal diagnosis Some families have a history of severe genetic disease, such as an affected previous child or near relative, may wish to determine the presence of the disorder in a developing fetus. Prenatal diagnosis allows for an informed reproductive choice if the fetus is affected. if the expected genetic abnormality produces a gross anatomic defect (e.g. neural tube defects), visualization of the fetus, for example, by ultrasound or fiberoptic devices (fetoscopy), will be quite useful. The biochemical analysis of the amniotic fluid can also provide diagnostic clues (e.g. presence of high levels of α-fetoprotein is associated with neural tube defects).

Karyotyping of the fetal cells (obtained either from the amniotic fluid or from biopsy of the chorionic villi) can be used to assesses the morphology of chromosomes. New staining and cell sorting techniques have permitted the rapid identification of trisomies and translocations that produce chromosomes of abnormal lengths. However, molecular analysis of fetal DNA promises to provide the most detailed genetic picture. The development of Polymerase Chain Reaction (PCR) has dramitically shortened the time needed for the DNA analysis.

The sources of DNA may be obtained from white blood cells, amniotic fluid, or chorionic villi.

Direct diagnosis of sickle cell disease using Restriction Fragment Length Polynorphism (RFLP): The genetic disorders of hemoglobin are the most common genetic diseases in humans. At present, treatment for most of these disorders is limited. Prenatal diagnosis (in association with genetic counseling) allows time for making informed decisions concerning continuation of the pregnancy. Sickle cell disease is an example of a genetic diseases caused by a point mutation.

In the past, Dx of Sickle cell anaemia involved the determination of the amount and kinds of hemoglobin synthesized in red cells obtained from fetal blood. For example, the presence of hemoglobin S in hemolysates indicated sickle cell anemia. However, the invasive procedures to obtain fetal blood have a high mortality rate (about 5%), and diagnosis cannot be carried out until late in the second trimester of pregnancy when Hb S begins to be produced.

By the technique of RFLP, diagnosis is more safe (because it takes fetal cells from either the amniotic fluid or the chorionic villi) and earlier. The sequence altered by the point mutation (A to T base mutation) abolishes the recognition site of the restriction endonuclease MstII that recognizes the nucleotide sequence CCTNAGG (where N is any nucleotide), Thus, the A-to-T mutation within a codon of the βS-globin gene eliminates a cleavage site for the enzyme

Normal DNA digested with MstII yields a 1. 15-kb fragment, whereas a 1 Normal DNA digested with MstII yields a 1.15-kb fragment, whereas a 1.35-kb fragment is generated from the βS gene as a result of the loss of one MstII cleavage site. Following electrophoresis, probes are used specifically for the β globin gene will yield different length fragments and thus settling the diagnosis of sickle cell disease (i.e. homozygus or SS) and sickle cell trait (heterozygus or AS) compared to normal (AA).

Indirect, prenatal diagnosis of phenylketonuria (PKU) using RFLP: Phenylalanine is an essential aromatic amino acid that is mainly (90%) metabolized in the liver by phenylalanine hydroxylase (PAH) enzyme to tyrosine. The gene for this enzyme that is located on chromosome no. 12 is deficient in PKU. This gene spans about 90 bp and contains 13 exons separated by introns. Mutations in this gene occur in all these exons mostly of missense type although splice, nonsense and silent mutations in addition to deletions and insertions can all occur.

How the gene will be identified? To establish a diagnostic protocol for this genetic disease, one has to analyze DNA of family members of the afflicted individual. The key is to identify markers (RFLP) that are tightly linked to the disease trait. Once these markers are identified, RFLP analysis can be used to carry out prenatal diagnosis.

DNA-based screening is useful not only in determining if an unborn fetus is affected, but also in detecting carriers of the mutated gene. PKU, like many inborn errors of amino acid metabolism, is inherited as an autosomal recessive trait. Identification of heterozygotes can aid in future family planning.