MB620 Bioinformatics at University of New Haven

MB620 Bioinformatics
University of New Haven
Instructor: Joel S. Bader
Class 12: By Popular Demand

Master Outline

Genetics
Traits/Genes to Location	Genetic and physical maps Research Genetics mapping panel Stanford mapping server
Traits/Genes to Experimental Organisms	Jackson Laboratories
Trait/Gene Location Database	OMIM, On-line Mendelian Inheritance in Man
Genomic DNA Analysis
Sequences to Contigs	CuraTools CAP, PHRAP
Contigs to mRNA	Genscan Grail tblastn (protein query, genomic database)
mRNA Analysis
DNA to Homolog	blastn, blastx, fasta
DNA to Protein	ORF finders NCBI ORF Finder
Protein Analysis
protein homologs	blastp
conserved residues	multiple sequence alignment, clustal-w
evolutionary history	Phylip, Paup
blastp for linguistics	AltaVista Babelfish
Prokaryot homologs	Clusters of Orthologous Groups
Domains	Pfam Prosite Prodom
Cellular localization	Psort
Secondary structure prediction	Consensus prediction
Tertiary structure prediction	Swiss-Model
Known folds	SCOP CATH, DALI
Structure similarity search	VAST
Bioinformatics Resources
GenBank, OMIM, Blast, Entrez	NCBI
Swiss-Prot, TrEMBL, Prosite	ExPASy

Primer design

Why design primers?

Is DNA present: Yes/No (what's a typical application?)
Amplify a particular region of DNA for later use

Amplifying DNA: Polymerase Chain Reaction (PCR)
(How was DNA amplified before PCR?)
Nobel Prize, Kerry Mullis

start: template + primers
heat: dsDNA -> ssDNA
anneal: ssDNA + free primer -> ssDNA-primer
extend: ssDNA-primer -> dsDNA
return to start

Requirements

Primers are specific. For human genome, log_4(3e9)= 16 nt. To be safe, 20-24 is typical range.
Primer are high concentration -> dimerization is possible. Screen out primer dimers.
One T(heat) for both primers. One T(anneal) for both primers. Adjust with GC content. How does this work?
Avoid known problems: repetitive elements (e.g. Alu), mis-hybridization.
Experimental requirements: position, reading frame, length for multiplexed assays, etc.

Visit CuraTools and use Primer3.

Steve Rozen, Helen J. Skaletsky (1996,1997)
   Primer3. Code available at
   http://www-genome.wi.mit.edu/genome_software/other/primer3.html

Codon usage

Remember ORF prediction: have mRNA, predict protein coding region
What does the protein coding region look like?
What is the proper reading frame?
Good hint: codon usage
Total number of codons: 64
Number of protein-coding codons: 63
Number of protein-coding codons used: less than 63, depends on species.
The codon usage database in Japan has codon usage information.

A little math

the entropy of a code S = - sum(words) prob(word) ln[prob(word)]
the effective number of words = exp(S)
if all N words have the same probability 1/N then
   the entropy = - sum(words) (1/N) ln(1/N) = ln(N)
   the effective number of words = exp[ln(N)] = N
if only one word is used then
   the entropy = - sum(words) prob(word) ln[prob(word)] = 0
   the effective number of words = exp[0] = 1

How do organisms stack up?

Organism	Effective number of codons
E. coli	51.8
S. cerevisiae	52.2
Chloroplast, Arabadopsis thaliana	54.2
Arabadopsis thaliana	55.1
Mitochondrion, Homo sapiens	46.8
Homo sapiens	54.7

Actually, it's not just codon usage that depends on organism.
The genetic code also varies.
See the genetic code page at NCBI.

Restriction enzyme sites

Visit CuraTools.
Use TACG to do simulated digests
TACG is Copyright (c) 1996,1997 by Harry Mangalam at Univ. of California, Irvine.

Medline

Journal database: PubMed (Medline) at NCBA
By now you're familiar with database searches.

New trends: Functional Genomics

From sequence to function.

Gene expression analysis

Expressed Sequence Tags (ESTs) (Incyte)
Serial Analysis of Gene Expression (SAGE) SAGE at NCBI
Differential Display, AFLP, GeneCalling (CuraGen)
Hybridization (Synteni/Incyte, Affymetrix)
Pat Brown's group at Stanford

Proteomics

Protein gels, 2D-polyacrylamide gel electrophoresis
Swiss-2DPage
Yeast two hybrid system (Stan Fields, Roger Brent, ...)

MB620 Bioinformatics University of New Haven Instructor: Joel S. Bader Class 12: By Popular Demand