There are a bunch of good PCR primer design programs on the web:
Primer 3 at the MIT Whitehead Institute
http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgiCassandra at the Univ. of Southern California
http://www-hto.usc.edu/software/procrustes/cassandra/cass_frm.htmlGeneFisher by Folker Meyer & Chris Schleiermacher at Bielefeld University, Germany
http://bibiserv.TechFak.Uni-Bielefeld.DE/genefisher/Xprimer at the Virtual Genome Center, Univ. Minnesota Medical School
http://alces.med.umn.edu/rawprimer.html
Mac/PC SoftwareThere are a number of (expensive) dedicated PCR primers design programs for personal computers that have “special features” such as nested and multiplex PCR :
Oligo (Molecular Biology Insights, Inc.)
Primer Premier (Premier Biosoft)
Many of the comprehensive MolBio. programs also have PCR features
MacVector
OMIGA
Vector NTI
GeneToolUsing Computers for DNA SequencingThe Biological Basis of DNA Sequencing Technology
Virtually all DNA sequencing, (both automated and manual) relies on the Sanger method
DNA replication with dideoxy chain termination
separation of the resulting molecules by polyacrylamide gel electrophoresis.
The DNA fragment to be sequenced must first be cloned into a vector (plasmid or lambda).
Then the cloned DNA must be copied in a test tube (in vitro ) by a DNA polymerase enzyme to obtain a sufficient quantity to be sequenced.
Limitations of the technologySequences can only be determined in approximately 400-800 base pair chunks known as “reads.”
This is due to both the biochemistry of the DNA polymerase enzyme and the resolution of polyacrylamide gel electrophoresis.
most genes contain many thousands of bp and many modern sequencing projects are intended to produce complete sequences of large genomic regions (millions of bp)
Assemby of ContigsAs a result, all sequencing projects must involve the division of the target DNA into a set of overlapping ~500 bp fragments.
and then the assembly of these fragments into complete sequences (contigs)
Contig = contiguous sequenced region
Assembly of overlapping fragments is a computational problem
Contig Assembly Problems1) The 500 bp reads of sequence data have errors of both incorrectly determined bases and insertions/deletions
2) The error rate is highest at the beginning and ends of the reads - precisely the regions that must be overlapped.
3) Some sequence from cloning vectors is often included at the ends of sequence reads
Sequence Assembly AlgorithmsDifferent than similarity searching
Look for ungapped overlaps at end of fragments
(method of Wilbur and Lipman, (SIAM J. Appl. Math. 44; 557-567, 1984)
High degree of identity over a short region
Want to exclude chance matches, but not be thrown off by sequencing errors
Vector removal uses similar approach, but less stringent
should recognize small regions of identity and tolerate more mismatches
Overlap at ends, not internalSoftware determines strategy
Based on their faith in the speed and reliability of sequence analysis/assembly software, researchers have generally taken one of three different approaches to planning sequencing projects:
Ordered sub-cloning
Primer walking
Shotgun sequencingOrdered cloningPeople who don't trust software generally put a lot of time into dividing large pieces of DNA into small ordered overlapping fragments
This strategy requires much more initial cloning work in the laboratory
but it minimizes the number of actual sequencing reads required to complete a project
It is easy to assemble the reads since it is known how they should fit together to form the final contig
Primer WalkingMake a new primer from the end of each new sequence read
It requires very fast and accurate analysis of sequence reads since each step uses information from the previous read
Skips sub-cloing step entirely since all sequencing reactions can be done on one large clone
上一篇：Polymerase Chain Reaction 聚合酶链式反应 下一篇：Real-time PCR实验注意事项
共5页: 上一页 [1] [2] 3 [4] [5] 下一页
