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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 limitationsUnderstand 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 www.firtsmarket.com/cutter
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: • Most stable• Longest extension
Analyzing DNA Composition
DNA composition varies a lotStability of a DNA sequence depends on its G+C
content (total guanine and cytosine)High G+C makes very stable DNA moleculesOnline 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/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/genomescan
Producing Genomic Data
Until recently, sequencing an entire genome was very expensive and difficult
Only major institutes could do itToday, scientists estimate that in 10 years, it will cost about
$1000 to sequence a human genomeWith sequencing so cheap, assembling your own genomes is
becoming an optionHow 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 piecesThen 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 www.phrap.org
Other programs exist for joining smaller datasets• For example, try CAP3 at pbil.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 Lab rulai.cshl.edu• EPD www.epd.isb-sib.ch