© Wiley Publishing All Rights Reserved. Working with a Single DNA Sequence

Learning Objectives Discover how to manipulate your DNA sequence on a computer, analyze its composition, predict its restriction map, and amplify it with PCR Find out about gene-prediction methods, their potential, and their limitations Understand how genomes and sequences and assembled

Outline 1.Cleaning your DNA of contaminants 2.Digesting your DNA in the computer 3.Finding protein-coding genes in your DNA sequence 4.Assembling a genome

Cleaning DNA Sequences In order to sequence genomes, DNA sequences are often cloned in a vector (plasmid, YAC, or cosmide) Sequences of the vector can be mixed with your DNA sequence Before working with your DNA sequence, you should always clean it with VecScreen

Computing a Restriction Map It is possible to cut DNA sequences using restriction enzymes Each type of restriction enzyme recognizes and cuts a different sequence: EcoR1: GAATTC BamH1: GGATCC There are more than 900 different restriction enzymes, each with a different specificity The restriction map is the list of all potential cleavage sites in a DNA molecule You can compile a restriction map with

Making PCR with a Computer Polymerase Chain Reaction (PCR) is a method for amplifying DNA PCR is used for many applications, including Gene cloning Forensic analysis Paternity tests PCR amplifies the DNA between two anchors These anchors are called the PCR primer

Designing PCR Primers PCR primes are typically 20 nucleotides long The primers must hybridize well with the DNA On biotools.umassmed.edu, find the best location for the primers:biotools.umassmed.edu Most stable Longest extension

Analyzing DNA Composition DNA composition varies a lot Stability of a DNA sequence depends on its G+C content (total guanine and cytosine) High G+C makes very stable DNA molecules Online resources are available to measure the GC content of your DNA sequence

Predicting Genes The most important analysis carried out on DNA sequences is gene prediction Gene prediction requires different methods for eukaryotes and prokaryotes Most gene-prediction methods use hidden Markov Models

Predicting Genes in Prokaryotic Genome In prokaryotes, protein-coding genes are uninterrupted No introns Predicting protein-coding genes in prokaryotes is considered a solved problem You can expect 99% accuracy

Finding Prokaryotic Genes with GeneMark GeneMark is the state of the art for microbial genomes GeneMark can Find short proteins Resolve overlapping genes Identify the best start codon GeneMark uses hidden Markov Models Use exon.gatech.edu/GeneMarkexon.gatech.edu/GeneMark

Predicting Eukaryotic Genes Eukaryotic genes (human, for example) are very hard to predict Precise and accurate eukaryotic gene prediction is still an open problem ENSEMBL contains 21,662 genes for the human genome There may well be more genes than that in the genome, as yet unpredicted You can expect 70% accuracy on the human genome with automatic methods Experimental information is still needed to predict eukaryotic genes

Finding Eukaryotic Genes with GenomeScan GenomeScan is the state of the art for eukaryotic genes GenomeScan works best with Long exons Genes with a low GC content GenomeScan uses Hidden Markov Models Homology searches It can incorporate experimental information Use genes.mit.edu/genomescangenes.mit.edu/genomescan

Producing Genomic Data Until recently, sequencing an entire genome was very expensive and difficult Only major institutes could do it Today, scientists estimate that in 10 years, it will cost about $1000 to sequence a human genome With sequencing so cheap, assembling your own genomes is becoming an option How could you do it?

Sequencing and Assembling a Genome (I) To sequence a genome, the first task is to cut it into many small, overlapping pieces Then clone each piece

Sequencing and Assembling a Genome (II) Each piece must be sequenced Sequencing machines cannot do an entire sequence at once They can only produce short sequences smaller than 1 Kb These pieces are called reads It is necessary to assemble the reads into contigs

Sequencing and Assembling a Genome (III) The most popular program for assembling reads is PHRAP Available at Other programs exist for joining smaller datasets For example, try CAP3 at pbil.univ-lyon1.fr/cap3.phppbil.univ-lyon1.fr/cap3.php

Going Farther Predicting when and how genes are expressed is one of the main challenges of modern biology It requires predicting genes It also requires predicting promoters The challenge is to find these regions and to understand the signals they contain Try the following resources: Zhang Labrulai.cshl.edu EPDwww. epd.isb-sib.ch