Mapping Of The Human Genome

Question 1

Genetic linkage maps

Answer 1

Don’t need to know sequence
Compared closeness based on inheritance of two genetic markers

Compares parental to recombinant type in F2 - less recombination by crossing over when genes are close

Units = centimorgans (relative positions)

Question 2

Physical map

Answer 2

Physical closeness determined by overlap in clones from a DNA library

Question 3

Steps for preparing DNA library

Answer 3

Cell lysis

DNA extraction

Partial digestion with restriction endonuclease

Join to vector
(Cut vector with restriction enzyme)

Selectively plate vector

Question 4

Important qualities of vectors

Answer 4

Must be able to take up plasmids and retain them quite well

Must be culturable

Need to be (-) for selective marker previous to plasmid transformation

Question 5

Disadvantages of YAC

Answer 5

Many chimeric clones (false overlap)

Clones are unstable -rearrangement and deletions occur

Difficult to remove YACs from yeast cell

Question 6

How do we re-order clones from libraries

Answer 6

Assess overlap- build contigs

1) make label with unique sequence - use sequence tag sites (STS)
2) PCT each clone with each STS

Statistically analyze overlap (STSs co-occur)

Arrange overlapping sections into contigs (should theoretically have 24)

Question 7

Making STS markers

Answer 7

Acquire random sequences from genome

Make primers that will amplify 200-500 bp

Test primers against whole genome

Markers that appear only once are good

Question 8

How do we map what are of the chromosome is represented by a clone or contig

Answer 8

FISH With same STS marker

Question 9

How can microsatellites be used as STSs

Answer 9

Primers pair to unique sequence flanking repeat

Question 10

Radiation hybrids

Answer 10

Way to clone long sections

Subject cultured human cells to irradiation

Fuse irradiated cells to hamster cells (20% of human DNA per clone)

Select for human/hamster hybrids

Screen with STS markers for overlap