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Dna Sequencing

Dna Sequencing



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Published by shailendra
DNA sequencing elucidated...
DNA sequencing elucidated...

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Published by: shailendra on Feb 21, 2009
Copyright:Attribution Non-commercial


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DNA Sequencing
CS262 – Lecture 9 NotesScribed by Dustin Chang2 February 2004
1DNA Sequencing Problem
The goal is to find the complete sequence of nucleotides (i.e. A, C, T, G) in a given sampleof DNA. Unfortunately, a machine that canreceive a long DNA sample as input andoutput its complete sequence does notcurrently exist. Current DNA sequencingtechnology can only directly sequenceapproximately 500 nucleotides at a time.
2Similarity of the Human Genome
In undertaking the monumental task of sequencing the entire human genome, decidingwhich particular individual's genome to sequence seems important. Although CraigVenter was the subject of Celera's sequencing effort, the actual genomic variation between individual humans is negligibly minimal. One hypothesis suspects that
 Homo sapiens
arose in Africa where a small population interbred, reducing overall geneticvariation. Dispersal from Africa of an even smaller subset of this populationapproximately 100,000 years ago across the rest of the world, further reducing geneticvariation.
Polymorphism rate
is defined as the number of nucleotide base changes between twodifferent members of a species. This occurs at an average rate of 1 in 1,000 bases in
 page 2 of 7humans; therefore, the nucleotide sequences of any two humans is roughly 99.9%identical. The polymorphism rate may be substantially higher in other species.Additionally, bases possessing the highest polymorphism rates are typically leastimportant, often falling in non-functional DNA where mutations in the nucleotidesequence have least impact on the organism's survival. Thus, similarity is preserved.
3Tools of DNA Sequencing
: small circular pieces of DNA.
Using restriction digest enzymes, the sample DNA is cleaved into shorter (~ 10
 bases) fragments. These restriction enzymes only cleave at specific recognitionsequences in the DNA. The vector is then cleaved using the same restrictionenzymes, allowing the DNA fragments (inserts) to incorporate into the vector.
VectorSize of Insert
Plasmid2,000-10,000(can control size)Cosmid40,000BAC (Bacterial Artificial Chromosome)70,000-300,000YAC (Yeast Artificial Chromosome)> 300,000(not used much recently)
Genomic DNA to be sequencedRestriction enzyme digestDNA fragmentsVector Circular genome(bacterium, plasmid)Knownlocation(restrictionsite)
 page 3 of 7
Gel Electrophoresis
: separation of DNA fragments by electric-field induced migrationthrough a gel matrix, which causes longer fragments to move more slowly than shorter fragments.
The vectors are now mixed with primers insolution. These primers will recognize therestriction sites marking the beginning andend points of the incorporated samplefragment and initiate synthesis of newDNA strands at those points.
During this synthesis reaction, one species(either A, C, T, or G) of fluorescentdideoxynucleoside is added to the reactionmixture of regular nucleosides. Anytime amodified nucleoside is added to a growingDNA strand, extension of that strand willhalt. This stops the synthesis reaction at all possible points.
The reaction products are then separated by gel electrophoresis. The resolution of gel electrophoresis decreases with increasing length of the DNA strands. This is the primary factor that limits the length of DNA that can be directly sequenced.
: output of gel electrophoresis that orders fragments by length anddistinguishes among terminating nucleotides of each fragment.
itor by Phil Green): popular dynamic programmingmethod used to read the sequence from an electropherogram following filtering,smoothing, and correction for length compressions.

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