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Genomics & Proteomics

• Genomics and proteomics studies are often used to analyze health diagnostics.
• The suffix "-omics" signifies the investigation of ample sets of biological molecules.
• Genomics is a sub-discipline of genetics covering:
  • Mapping
  • Sequencing
  • Functional analysis of genomes.
• Genomics focuses on DNA sequences, genome organization, function, and evolution.
• Genomics attempts genome-scale analyses for genetic patterns within a species.
• Genomics deals with systematic genome data to answer questions in biology, medicine, and industry.

Omics Cascade

• Genomics analyzes the genome and gene structure, through DNA sequence analysis, gene expression analysis, gene and promoter field cloning and characterization, and molecular markers.
• Proteomics focuses on characterizing genome functioning at protein levels, involving recombinant gene expressions.
• Metabolomics analyzes metabolic profiles and status, by means identification and characterization of metabolite synthesis and accumulation.
• Bioinformatics integrates data obtained from the other omics studies.

Proteomics Defined

• Proteomics aims to identify all proteins in a cell or organism and their
  • Posttranslational modifications
  • Cellular localization
  • Functions
  • Interactions
• Proteomics studies the proteome, the entire complement of proteins encoded in a genome.
• Protein chemistry deals with individual proteins, complete sequence analysis, emphasis on structure and function, and structural biology.
• Proteomics deals with complex mixtures, partial sequence analysis, emphasis on identification by database matching and in systems biology.

Genome Types

• Prokaryotic genomes are one of the types of genomes
• Eukaryotic Genomes are also a type of genomes, comprising:
  • Nuclear Genomes
  • Mitochondrial Genomes
  • Chloroplast Genomes

Gene Evolution and Variation

• During evolution, genes may pass to descendants, accrue mutations, be lost, duplicated (followed by divergence or loss), or undergo horizontal transfer.
• Homologues are genome regions or proteins derived from a common ancestor.
• Paralogues are related genes in the same species that have diverged to provide separation of function.
  • Alpha and beta chains of haemoglobin are paralogues.
• Orthologues are homologues that perform the same function in different species. -Human and horse myoglobins are orthologues.

Genome Sequencing Factors

• Genome sequencing finds the complete DNA sequence of an organism for insights into disease, species relationships, and gene function.
• This sequencing has led to development such as genomics, proteomics, and metabolomics.
• Sequencing is easier in prokaryotic organisms because their genomes are smaller with fewer repetitive sequences.
• Eukaryotic genome sequencing proves more difficult because of their larger size and more repetitive sequences.
• Technological advances and computerized data handling have facilitated sequencing for large genomes like the human genome.
• Nucleotide sequences are high-resolution maps providing gene information, regulatory elements and genome features.

Genome Sequencing Methods

• Eukaryotic genome sequencing is difficult because of its size and repetitive sequences.
• Improvements in technology and computerized data handling have opened sequencing for even very large genomes like the human genome.
• Automated sequencing techniques were developed and executed after the Human Genome Project.

Hierarchical Shotgun Sequencing/Clone-by-Clone Sequencing

• Can generate high density maps, making the genome assembly easier.
• Time-consuming and costly so it is seldom used at present.
• Initially adopted by the HGP consortium
• Comprises four steps:
  • Preparation of BAC clone library
  • Preparation of clone fingerprint
  • BAC clone sequencing
  • Sequence assembly

Whole Genome Shotgun Sequencing (WGS)

• Generally involves six steps:
  • Isolation of genomic DNA
  • Random fragmentation of genomic DNA
  • Size selection using electrophoresis
  • Library construction
  • Paired-end sequencing (PE sequencing)
  • Genome assembly
• Two different sizes of DNA fragments are used, including longer insert (2-2.5 kb) and short insert (0.5-1.2 kb), selected from the agarose gel.
• Long inserts are cloned in phage or cosmid vectors, while short inserts are cloned in plasmid vectors.
• The short insert clone library is used for sequencing from both ends.
• Since large numbers of clones are sequenced, each of the genomes will be covered more than 10 times.
• Long insert clones can be used to increase the efficiency of genome assembly.

DNA Sequencing

• Sanger DNA sequencing works for a distance beyond the sequencing primer, best from 30 nt to 350 nt
• Products beyond that distance are reduced because chain termination has already occurred.
• Shotgun sequencing is used to sequence very large amounts of DNA, including entire genomes.
• In shotgun sequencing, DNA is shredded into smaller fragments for individual sequencing.
• The sequences of these fragments are then reassembled into their original orders.
• DNA shredding can be done via restriction enzymes or mechanically by shearing.
• The alignment of overlapping pieces is done via computer.
• Some vectors for sequencing include:
  • Plasmids for E. coli hosting, up to 15kb inserts
  • Phage for E. coli hosting, up to 25kb inserts
  • Cosmids for E. coli hosting, up to 45kb inserts
  • BAC for E. coli hosting, 100-500kb inserts
  • YAC for Yeast hosting 250-1000kb inserts
• Contigs, are groups of overlapping pieces of chromosomal DNA.
  • They are created by making contiguous clones
  • The contig is the smallest number of inserts that covers a region of the chromosome.
• Paired-End Sequencing involves short-insert paired-end reads.
• Mate Pair Sequencing involves genomic DNA fragmentation with fragments between 2 and 5 kb. Circularize, then do enrich Biotinylated Fragments. Then fragmentation (200-600 bp. and finally the Ligate Adapters

Sanger sequencing

• Called the “chain termination method" which is for determining the nucleotide sequence of DNA.
• Developed by two time Nobel Laureate Frederick Sanger and his colleagues in 1977
• The Dideoxynucleotide method is the most common method employed today.
• Steps in Maxam-Gilbert sequencing: -Label 5'- end of DNA -Aliquot DNA sample in 4 tubes -Perform base modification reaction -Perform Cleavage reaction -Perform Gel Electrophoresis -Perform Autoradiography -Interpret results
• Labeled DNA fragment is divided into four aliquots each treated with reagent modifying a specific base
  • Aliquot A + dimethyl sulphate, methylates guanine
  • Aliquot B + formic acid, modifies adenine and guanine
  • Aliquot C + Hydrazine, modifies thymine + cytosine
  • Aliquot D + Hydrazine + 5 mol/l NaCl makes for reaction specific to cytosine
• These four are incubated with piperidine, to cleave sugar phosphate backbone of the DNA next to residue was modified.
• Sanger dideoxy sequencing uses -Isolate DNA -Fragmentation using hydroshear -Aliquot -Add adaptors -Add primers+polymerase+dNTPs+ddNTPs+Magnesium -Run PCR -Polyacrylaminde gel electrophoresis -Deduce the sequence -Write in 5' to 3' the complementary strand and reverse it. That is your original strand in 5' to 3'

Sanger Sequencing vs. Fluorescence DNA Sequencing

• Sanger uses the radioactive labelled
• Fluorescence DNA uses fluorescent molecules are being used instead of radiolabelled, like Fluorescein and Rhodamine derivates covalently attached to nucleotides
• Automated sequencing utilizes automatic sequencers.
• Disadvantages:
  • Good only for 500-750bp reactions
  • Time consuming
  • The human genome is about 3 billion bp.
• "N" designation, represent peaks of different colors instead of just one
• Example, Phred base-calling which widely used software program