Lecture 26 Flashcards
What is the genome size and number of genes in E. Coli?
4.7 Mb, 4288 genes
What is the genome size of yeast (Saccharomyces Cerevisiae) and number of genes?
13 Mb and 6241 genes
What is the genome size of Caenorhabditis elegans (‘worm’) and number of genes?
140 Mb and 18, 428
What is the genome size of Drosophila melanogaster (‘fly’) and number of genes?
120 Mb and 13, 601
What is the genome size of Arabidopsis thaliana (‘weed’) and number of genes?
125 Mb and 26000 genes
What is the genome size of Homo sapiens (man) and number of genes?
3.2 Gb and 21,000 genes
How is the catch 22 of needing to know some sequence before cloning can be done overcome?
The sequence to be determined can be ligated to a known sequence allowing there to be universal primers which will anneal to adjacent multiple cloning sites of common vectors
What is the sanger method of sequence determination?
dideoxy chain termination, with fluorescent labelling which is then read by a computer
What are the limitations of using the sanger method to determine DNA sequence?
The initial sequence close to the primer is hard to read, the next few 100 bases will have good resolution further out from this there will be diffusion of bands and less consistent incorporation of dideoxy nucleotides making it much harder to read
What technology has advanced the efficiency of the sanger method of sequencing?
Capillary automated loading of gels rather than manual loading
How does pyrosequencing determine DNA sequence?
A sample DNA has each of the bases added and then washed off in turn, there is an enzymatically generated flash of light each time a new base is added so the order can be determined allows 4-500 bases per run
How is the flash of light generated in pyrosequencing?
When a nucleotide is added a pyrophosphate is generated this is then acted on by sulfurylase to form ATP
Luciferase then uses this ATP to produce light
Apyrase is the enzyme used to ‘clean up’
What is the Roche Sequencer?
Based off the same principal as pyrosequencing, this has a much higher throughput due to massively parallel reactions and gives reads of 500 bp
What is the flaw with both Roche and Pyrosequencing methods?
There is a high rate of error if more than one of the same base is added (due to it being repeated in the sequence), if there are only a few repeats then this may be detected by a change in the intensity of the light, but as the number of repeats increases so does the error rate
How is complete sequence data obtained from multiple random, shorter sequences?
A random library is constructed with 5-20 times genome coverage
Each clone is sequenced and then these sequences are put in order by a computer which identifies sequence overlap to place them into contigs
