MBB 141 Questions 24-26

24. YAC clone contigs can be assembled following STS mapping. First, the locations of STSs on YAC clones are mapped using PCR. Then YAC clones that share STSs are aligned. The first step usually involves repeated screening of a library of YAC clones with different STS markers. In practice, some of the STSs may be well characterized and even localized to specific chromosomal regions or genes, whereas others may be less well characterized. In a typical screen of a YAC library constructed from the genome of a higher eukaryote, most STSs identify only a few YACs. However, a few STSs identify dozens of YACs. What might be the basis of this difference? How does the difference influence the assembly of a contig?

25. The average size of fragments, in base pairs, observed after genomic DNA from 8 different species was individually cleaved with each of 6 different restriction enzymes, is shown below:

Species	ApaI	HindIII	SacI	SspI	SrfI	NotI
	GGGCCC	AAGCTT	GAGCTC	AATATT	GCCCGGGC	GCGGCCGC
Escherichia coli	68000	8000	31000	2000	120000	200000
M. tuberculosis	2000	18000	4000	32000	10000	4000
S. cerevisiae	15000	3000	8000	1000	570000	290000
A. thaliana	52000	2000	5000	1000	No sites	610000
C. elegans	38000	3000	5000	800	1.11M	260000
D. melanogaster	13000	3000	6000	900	170000	83000
Mus musculus	5000	3000	3000	3000	120000	120000
Homo sapiens	5000	4000	5000	1000	120000	260000

continuing the table...

Species	NotI
	GCGGCCGC
Escherichia coli	200000
M. tuberculosis	4000
S. cerevisiae	290000
A. thaliana	610000
C. elegans	260000
D. melanogaster	83000
Mus musculus	120000
Homo sapiens	260000

a) Under the assumption that each genome has equal amounts of A, T, G, and C, and that on average these bases are evenly distributed, what average fragment size is expected following digestion with each enzyme?

b) How might you explain each of the following?

i. There is a large variation in the average fragment sizes when different genomes are cut with the same enzyme.
ii. There is a large variation in the average fragment sizes when the same genome is cut with different enzymes that recognize sites having the same length (e.g., ApaI, HindIII, SacI, and SspI).
iii. Both SrfI and NotI, which each recognize an 8-bp site, cut the Mycobacterium genome more frequently than SspI and HindIII, which each recognize a 6-bp site.

c) Based on this data, which enzymes would be good choices for constructing a restriction map of a chromosome (or a large segment of a chromosome) in each organism? Explain your choices.

26. STS mapping has been useful to generate clone contig maps. Perform the following exercise to consider the logistics of locating STSs using PCR.

a) A plasmid library contains 500-bp inserts generated from randomly sheared mouse DNA. How would you identify clones harboring STRs with the dinucleotide repeat (AT)_N or the trinucleotide repeat (CAG)_N?

b) How would you use these STRs as STSs in a mapping experiment?

c) Nusbaum and colleagues generated a YAC-based physical map of the mouse genome by localizing 8,203 STSs onto 960 YAC clones. If each of the STSs were assayed in each of the 960 YAC clones, how many different PCRs would need to be analyzed?

d) Although PCRs can be performed robotically, each reaction consumes time and material resources, and with each there is a certain chance of a false positive result or other error. It is therefore advantageous to reduce the number of PCR reactions by pooling individual YACs together. First, yeast colonies containing the YACs are grown individually in the wells of ten 96-well plates. Suppose the plates are numbered I to X and the wells of each plate are arrayed in an 8-row x 12 grid. The rows are coded A-H and the columns coded 1-12. This allows the position of a single YAC to be specified uniquely by a code (e.g., II-C6 specifies the YAC clone from plate II, row C, column 6). In one pooling scheme, all YACs from each row of a plate are pooled into one well (e.g., those on plate II, A1-A12 are pooled together into a well designated II-A) and all YACs from each column of a plate are pooled into one well (those on plate II, A7-H7 are pooled together into a well designated II-7).

i. How many different pools would now have to be screened with each STS?
ii. How many PCRs would have to be performed?
iii. If the II6 and IIF pools had a positive result with STS #6239, what is the code of the YAC containing this STS?
iv. How would you interpret a result where only the IV3 pool was positive for a particular STS?

e) Construct a YAC contig based on the results in the following table

STS marker	Positive YAC pools
63	II-6, II-A
210	II-6, II-A, IV-C, IV-3
522	VII-E, VII-12, X-G, I-C, I-8
713	I-C, I-8
714	VII-E, VII-12
719	X-H, X-9, IV-C, IV-3
991	X-H, X-9, VII-E, VII-12
1071	II-6, II-A, IV-C, IV-3, X-H, X-9
2631	II-6, II-A
3097	VII-E, VII-12, I-C, I-8
4630	VII-E, VII-12, I-C, I-8
5192	X-H, X-9, IV-C, IV-3
6193	X-H, X-9, VII-E, VII-12
6892	II-6, II-A, IV-C, IV-3

f) Devise a method to combine the YACs pooled in (c) to further reduce the number of PCRs. In your method, how many pools are there and how many PCRs must be performed?