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GENOMICS and BIOINFORMATICS 

Introduction

There are several related "advanced" areas in molecular biology:

• Genomics
• Post-genomics or functional genomics
• Bioinformatics

 

Here's how these areas relate to each other:

Genomics

• a broad term describing the acquisition of sequence data
•  many individual sequences of 500-800bp must be assembled
  n

Post-genomics or functional genomics

• nthe analysis of a genome sequence to locate genes, control sequences, etc
• nexperiments to determine the functions of unknown genes

Bioinformatics

• nthe use of computer systems to aid genomics and post-genomics research
• ncomputerized assembly of sequences, nexamination of sequences for the presence of genes, the nprediction of gene function and the nstorage of vast amount of data

Developmental Timeline:

http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture7/a1GenomeHistory.jpg

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n

Genomics - How to Sequence a Genome

 

 

1. The “Shotgun” approach to genome sequencing:

• ngenome is randomly broken into short fragments
• nidentify overlaps  
• nmust be accurate and unambiguous
• successful mainly with smaller bacterial genomes (without repetitive sequences)

        Problems with Shotgun Cloning:

• nsuccessful when no repetitive DNA is present
• nrepetitive DNA causes problems
• nparts of the genome may be assembled in the incorrect position or left out entirely

2. The "Clone Contig" Approach

• nconstruct overlapping series of cloned DNA fragments, then shotgun method for the fragments
• nthe cloned fragments should be as long as possible
• ncan provide an accurate sequence of a large genome that contains repetitive DNA
• nneeds much more work, time and money - can be slow, laborious

  Need to find the matching overlaps, and the software definitely helps!

  

3. Rapid Methods for Clone Contig Assembly

a. Clone fingerprinting

• nbased on the identification of sequence features that are shared by a pair of clones
• nrestriction digestion à look for clones sharing restriction fragments of the same size
• ninaccurate, laborious

b. Clone Contig assembly by “Sequence Tagged Site" (STS)

• n

  search for pairs of clones that contain a specific DNA sequence that occurs at just one position in the genome à they overlap

• nsequence tagged site (STS)
• noften part of a gene that has been sequenced in earlier project (does not have to be a gene)
• na pair of PCR primers can be designed

http://dean.pku.edu.cn/jiaoxue/zhubmb/chapter9/wpe40.jpg

 

Using a Map to Aid Clone Contig Assembly

Genetic Maps

 

a.  Obtained by genetic studies using Mendelian principles; using:

 

• ndirected breeding or pedigree analysis
• nany genetic marker can be used for mapping
• nRestriction fragment length polymorphisms (the catch: 100,000 RFLPs in the human genome

b. Short tandem repeats (STRs,  microsatellites)

• n

  made up of short repetitive sequences

• nPCR then agarose or polyacrylamide gel electrophoresis
• nat least 650,000 STRs in the human genome

  

  http://med.kuleuven.be/labfor/eng/_img/identification6.png

c. Single nucleotide polymorphisms (SNPs)

• npositions in a genome where any one of two or more different nucleotides can occur (point mutations)
• ntyped with short oligonucleotide probes
• nat least 1.4 million in the human genome

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Physical Maps of a Genome

• ndirectly locate the positions of specific sequences on chromosomal DNA (map is very important when a larger genome is sequenced (provides a guide)
• nany DNA marker is OK (often short sequences obtained from the ends of cDNAs)

nThe Tools:

• n

  direct examination of chromosomal DNA

• nfluorescent in situ hybridization (FISH), high degree of accuracy
• nmapping reagent
• na collection of overlapping DNA fragments spanning the chromosome or genome radiation hybrids

n

http://www.chrombios.com/WebFinals/AboutFISH/Bilder150dpi/8CGHSchema.jpg

 

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Post Genomics

• nTrying to understand a Genome Sequence - what does all this information mean?
• nneed to locate all the genes & determine their functions, but it is challenging!
• n2400 out of 6000 genes of S. cerevisiae are still orphans (no assigned functions)

Identifying the Genes in a Genome Sequence

• n

  Searching for open reading frames (ORFs)

• nORF: a series of nucleotide triplets beginning with an initiation codon and ending in a termination codon
• nlooking for ORF is the first step in gene location
• nall reading frames need to be searched
• nit is much more difficult in eukaryotes
• nin yeast over 400 short ORFs in questionable category

http://asianka.w.interia.pl/grafiki/jadro/gen.jpg  

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BIOINFORMATICS

http://www.stat.purdue.edu/images/bioinformatics/Bioinformatics_doerge_080304%20(1)-sm.jpg

Homology Search

• naccessed through the internet, BLAST (Basic Local Alignment Search Tool) online database is used to compare sequences and search for gene functions
• nif the test sequence is over 200 amino acids in length and has 30% or greater identity with a sequence in the database, then the two are almost certainly homologous
• nconfirmation is conducted by transcript analysis

http://bioinformatics.org/annhyb/images/fasta_blast_scan.png

 

Determining the Function of an Unknown Gene

• norphans are genes / sequences (not located / determined)
• nbioinformatics (the use of mathematical models and online search tools
• nsecondary structure prediction using computer programs
• nmembrane spanning motifs, DNA binding motifs, etc
• ngene knockout using “knockout mice”
• nsome knockouts have no obvious effect (either dispensable or too subtle to be detected)

  

Knockout Mice

• nKnockout mice contain the same, artificially introduced mutation in every cell, “knocking out” the activity of a pre-selected gene.
• nThe resulting mutant phenotype (appearance, biochemical characteristics, behaviour etc.) may provide some indication of the gene’s normal role in the mouse, and by extrapolation, in human beings.

http://www.bioteach.ubc.ca/CellBiology/StudyingGeneFunction/Chimeric%20mouse.gif

 

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