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Genbank Impact on Modern Molecular Biology Research
Molecular biology, along with GenBank plays an important role in genetic disorders. More than 200 single-gene disorders have been detected. It determines the molecular genetic defects in various diseases. These include amongst others cystic fibrosis, sickle cell anemia, and thalassaemia. Modes of inheritance are essential knowledge in genetic counseling and antenatal diagnosis.
Interest is fast increasing to determine nucleotide sequences. Articles are appearing to determine the sequences. Gen Bank and various journals are trying hard to publish nucleotide sequence data. Briefly, the nucleus synthesizes messenger RNA (mRNA). It conveys genetic information in code through the pores in the nucleus. mRNA is formed in DNA strands. In addition, DNA directs the synthesis of specific mRNA. The mRNA coming out of the nucleus conveys the DNA message to the protein-synthesizing center (ribosome) of the cytoplasm. It is attached to the surface of the ribosome to direct the ein synthesis. GenBank is the fundamental element of Molecular biology. It is the sequence formed and stored in the database of the entire genome of life.
Molecular basis of genetics
Deoxyribonucleic acid (DNA) is the storehouse of genetic information. It is a polymer consisting of two strands. Theses two wounds other. Thus a helix is formed. Every single strand consists of a basic unit called nucleotides. On the other hand, nucleotides are composed of three very important parts namely, a pentose sugar molecule known as 2-deoxyribose, a phosphate group, and a nitrogenous base. There are four different nitrogenous bases. These can be further classified into the pyrimidines cytosine (C) and thymine (T) and the other are purines (adenine (A) and guanine (G).
Phosphodiester bonds join the nucleotides into polynucleotide strands. When two of the strands wound up, they form the double helix of DNA. The phosphatee group of one nucleotide and the 5 carbon of the deoxyribose form phosphodiester. Thus, phosphonucleotide has a sugar-phosphate backbone. This sugar-phosphate backbone has a 5 end and a 3 end. The two strands of DNA are held up by the hydrogen bonds between the bases. The length of the DNA is measured by number of base pairs (bp). DNA 1000 bp long is one kilobase pair (km) in length. (Tanaka, 101-113)
Genes
A gene is a portion of a DNA molecule. Genes instruct formation of specific proteins. Number of genes in human beings varies from 30.000 to 100,000 in number. Size of genes varies greatly. The enzymatic function of the cell can decipher one of the many polynucleotide chains of DNA. It can verify particular sequence of a base pair, which constitute a gene. Gene for the muscle protein known as dystrophin may contain millions of base pairs.
Transcription
There is a particular order in a polynucleotide strand in recognizing short DNA sequences of As, Cs, Gs and T3. This particular order differ them from the other. This is the upstream sequence of the gene and is demarcated as the promoter sequence. There are different promoters in different genes. RNA polymerase, an enzyme, pinpoints the DNA sequences in the promoter site. When the sequence TATAAAT is recognized, it binds into the DNA. This is described as the TATA box. This is situated about 35 bp upstream, the site where transcription begins. The RNA polymerase transcribes the DNA sequence. Thus, it becomes single stranded molecule known as ribonucleic acid (RNA). The RNA polymerase follows the route along the strand. It stops transcribing when it reaches the end of the gene.
RNA splicing
Splicing follows transcription in the nucleus of the cell. Enzymes cut the coding sequences and subsequently spliced together. Messenger RNA is thus formed. Then it finally passes into the cytoplasm.
Translation
Organelles ribosomes in the cytoplasm can distinguish RNA sequence. They prepare amino acids in the polypeptide chain. This is encoded by the spliced mRNA. The sequence in the nucleotides is later translated into a polypeptide.
A ribosome is linked to the mRNA. The first nucleotides are possibly regulatory sequences. The middle region of the mRNA is the coding region. These extend over thousands of base pairs. The nucleosides at the end of mRNA are mostly of regulatory sequences.
The nucleotides in the coding region remain as sets of three. Three attached nucleotides, as a group is known as codon. Each codon acts as a specific instructor for an amino acid. It has two instructions for the amino acids. An amino acid may be added to a peptide chain or it may stop the chain. For these instructions, this is known as genetic code. Small RNA molecules in the cytoplasm carry amino acids in the polypeptide chain. These are known as transfer RNA (tRNA). Each tRNA has specific function for one amino acid. It has also three unpaired nucleotide bases. This is anticodon. It has its complimentary codon in the mRNA. The tRNAs carry specific amino acid to the codons of the mRNA. However, there are some other codones, which do not have coding functions for amino acids such as UAA, UAG and UGA. These are stop codones. Translation stops with these codones if ribosome finds one such codone. No amino acids are added to the polypeptide chain. Finally, ribosome and mRNA drift apart.
Mutation
DNA replication is a very intricate and accurate process. Still aberration occurs. These produce changes or mutations. Various factors are responsible for it. These can be enumerated as ultra violet light, radiation or chemicals. Transcription and translation affects the gene by changes in the amino acid sequence in the protein, which remains confined within the gene. However, in some cases protein functions are maintained yet some may cease or absolutely change. These lead to clinical disorder. Mutations are of different types.
Point mutation
When one nucleotide is substituted for another codone, it changes in a coding sequence. This is the simplest type of change. Lysine changes to arginine when AAA is mutated to AGA. When a critical part of the produced protein is changed, a grave clical disorder follows. Generally, substitution do not always seriously affect the function or stability of the protein produced. However, several codones act for the same amino acid. Nevertheless, mutation in beta-globine gene produces sickle cell disease. Valine is incorporated instead of glutamic acid within the poypeptide chain.
Insertion or deletion
Serious changes are found when there is insertion or deletion in one or more bases. With insertion of one nucleotide or deletion of one nucleotide, the sequence is changed. Different amino acids enter the polypeptide chain. Such changes are responsible for thalassaemia of some forms. Many hundred pairs of base pairs of DNA can be involved by insertions or deletions.
Splicing mutations
Abnormal splicing mutations may change intron sequences. It alters the amino acids incorporated within the poypeptide chain.
Termination mutations
When the ribosomes, which process the mRNA, stop codones, normal polypeptide chain terminates. Premature termination or late termination will result in mutations involving these codons.
Techniques for DNA analysis and isolation of genes
Use of recombinant DNA has led to in-depth study of functioning of genes and the pathology of diseases arising out of genetic disorders. Preparation of genomic DNA is the first step in studying the DNA of an individual. 20 ml of blood is drawn. Lymphocytes are made to break open the nuclear membrane and cell membrane too. Chromosomal DNA is extracted chemically. As DNA is very stable it can be stored frozen for many years.
Restriction enzymes cut genomic DNA into many fragments. These enzymes are obtained from bacteria. Recognition of specific DNA sequences is done by these enzymes. They also cut double-stranded DNA at these sites. Human genomic DNA is cut into huge number of fragments. Size and charge of DNA can be determined by electrophoresing the DNA by a gel matrix. Molecular size could be distinguished and it helps in long-range mapping of the genome to diagnose major deletions and rearrangements. (Kameyama & Nakagoshi, 205-216)
Conclusion
Southern blotting and DNA probes visualize individual DNA fragments..Presence or position of a particular gene can be settled using a gene probe. Blotting RNA fragments is known as Northern blotting and blotting proteins is called Western blotting.
With in a few hours the polymerase chain reaction can amplify over a million times minute amounts of DNA. The exact DNA sequence, which has to be amplified, has to be known before hand. Polymerase chain reaction allows millions of amplifications. Genetic research has thus bean revolutionized by this technique. Buccal cell scrapings, blood spots or single embryonic cells can be amplified. DNA cloning can be done by isolating and inserting a DNA fragment into a vector.
References
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Kameyama, Yoshiaki, & Nakagoshi, N. Patterns and levels of gene flow Molecular Ecology 10.1 (2008): 205-216.
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Tanaka, Seiji, Systematic mapping of autonomously replicating sequences Yeast 12.2 (2007): 101-113.
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