Humana

4.1.Introduction

4.1.1 Historical background:

The discovery of DNA structure by Watson and Crick, 1953, lead to a revolution in the study of biology. The DNA discovery enabled us to decode the genomic information for life that is contained within every dividing cell. With the advances in computer

technology we became able to store and process massive amounts of nucleotide sequence information. The combination of advances in biotechnology and

bioinformatics has provided us with the ability to study the biology of thousands of genes at the same time. Unfortunately, sequencing billions of bases of DNA does not tell us the function of all genes, how cells work or what goes wrong in a disease (Lockhart & Winzeler, 2000).

Expressed sequence tags (BSTs) are leading the way in gene discovery, and gene expression analysis. BSTs are partially sequenced cDNAs synthesised from randomly selected gene transcripts. Once created, the partially sequenced tags can be stored electronically and analysed. BSTs, once in hand, represent a sequence-based link from which each gene can be probed and monitored.

The importance of BSTs is derived from the fact that in any cell, only a subset of its genome is actively transcribed, known as the transcriptome. Isolating mRNA and constructing a cDNA library will make a collection of cDNA molecules that represent the subset of genes in use by that cell. A digital image of gene transcription levels for a particular cell line or tissue can be obtained by counting the number of BSTs in a random sampling that matched a given gene. Recently, with more sequence data

obtained, the sequence database has emerged where sequence homology, gene function, and gene expression data are correlated (Zweiger & Scott, 1997).

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The most fundamental application of EST databases is finding gene family members such as the chemokine family (Wells & Peitsch, 1997) and the tumour necrosis factor alpha (TNF a) receptor families (Wiley et al., 1995).

Other applications include detecting gene expression levels particularly genes whose expression levels change in diseased versus non-diseased tissues. With multiple transcripts obtained from a large set of tissues, statistically significant correlation may be obtained providing information about disease states, treatment outcome, or

genotypes (Spanakis & Brouty-Boye, 1997).

4.1.2.Computer analysis of newly identified cDNAs: 4.I.2.I. Basic Local Alignment Search Tool (BLAST):

BLAST is the search algorithm used by the programmes blastp, blastn, blastx, tblastn, and tblastx. The BLAST programmes are designed to search sequence similarity and identify homologues to a query sequence. Different BLAST programs can perform different tasks as follows:

blastn compares a nucleotide query sequence against a nucleotide sequence database. blastp compares an amino acid query sequence against a protein sequence database, blastx compares the translation products of a nucleotide query sequence on both strands against a protein sequence database.

tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames on both strands.

tblastx compares the six-ffame translations of a nucleotide query sequence against the six-ffame translations of a nucleotide sequence database.

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Search strategy:

The unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). HSP consists of two sequences, one from the query sequence and one from a database sequence, of arbitrary length whose alignment is maximal. Only matches that satisfy a threshold of significance are reported.

4.1.2.2. NIX tool:

NIX is a WWW tool used to analyse a chosen DNA sequence using many DNA programs including GRAIL, Fex, Hexon, MZEP, Genemark, Genefmder, Fgene, Blast, Polyah, RepeatMasker, and tRNAscan. NIX is designed to help the identification of genomic nucleic acid sequences and find predicted exons.

4.1.3. Amplification of complete cDNA molecules: 4.1.3.1. 5 -SMART RACE cDNA Amplification:

The method of SMART RACE cDNA amplification performs both 5’- and 3’-

amplification of cDNA ends (RACE). The SMART (Switching Mechanism At 5’ end of RNA Transcript) cDNA synthesis technology allows one to isolate the complete 5’ sequence of the target transcript. It allows the use of first-strand cDNA directly in RACE PCR without the need for adaptor ligation. Full-length cDNAs can be generated in reverse transcription reaction by the joint action of the SMART oligonucleotide and MMLV reverse transcriptase (RT). Certain MMLV-RT exhibit a terminal transferase activity that adds 3-5 residues , predominantly dC, to the 3’- end of the first strand cDNA when they reach the end of an RNA template. The SMART oligo is a stretch of dG residues that anneal to the dC rich cDNA tail and serve as a template for RT. A complete cDNA copy of the original RNA is thus synthesised with the addition of SMART sequence at the end. The dC-tailing of RT is efficient only when the enzyme

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reaches the end of the RNA template, thus the SMART sequence is added only to complete first-strand cDNAs. The first-strand cDNA is then used directly in 5’- and 3’- RACE PCR reactions without the need for second-strand synthesis.

The requirement for SMART RACE cDNA amplification is sequence information of about 23-28 nucleotides in order to design gene specific primers to be used with the universal primers. This limited requirement makes the method ideal for characterising genes identified through different methods including cDNA subtraction, differential display, RNA fingerprinting and library screening.

4.1.3.2. Inverse PCR:

The principle of this method is to generate a double stranded cDNA that starts with a known sequence followed by the unknown target sequence. The cDNA is then digested with a restriction enzyme which usually generates a 2-3 kb fragment containing a segment of the target sequence. The DNA fragment is then circularised by ligation. The circular DNA may be relinearised with a restriction enzyme that cleaves once within the target sequence. DNA is denatured and annealed with oligonucleotide primers that have been selected complementary to sequences at the 5’-end of target DNA followed by cycles of amplification in PCR (Ochman et al., 1988). The product of amplification reaction consists of a head-to-tail arrangement of the sequences that flanked the target region (figure 4.1).

4.1.4. Multiple tissue expression study:

One of the most powerful tools of studying sequence information is the use of high- density arrays of oligonucleotides or cDNAs. Nucleic acid arrays work by hybridisation of labelled RNA or DNA to nucleic acid molecules attached onto a surface.

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Hybridisation of a chosen sample to an array simply means a search by each labelled molecule for a “matching partner” on the surface.

One of the most significant applications for arrays is studying the patterns of gene expression, mRNA abundance, in different disease situations and in different tissues. This expression profile is a major determinant of cellular function as this profile changes with different situations.