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restriction enzyme fingerprinting

BAC clones can also be ordered by restriction enzyme fingerprinting. A typical BAC may be around 200kb and EcoRI may cut on average roughly once every 4kb. Hence, a typical BAC EcoRI digest may contain 40-50 bands. The accuracy with which band sizes can be measured is very important for this type of mapping. It is likely better than to the nearest 50bp but for convenience in this question let us assume that recording a band (on a computer file) as 2.1kb means it could be anywhere from 2,050bp to 2,149bp. Also, band intensities are measured, so if a particular size (say 3.2kb) has a band that is three times as strong as expected it is recorded as 3.2 x 3.

(i). The first step here is not so obvious. As a first idea (not necessarily a good one) I will pick out a particular signature of EcoRI band sizes (15.1, 8.3, 4.3, 2.3, 1.7kb) and pay attention only to BACs that include that signature. That signature will be quite common (because size resolution is not exact), so among 200,000 BAC clones (roughly the number likely to be used) there would likely be a few hundred with that signature. I will therefore list the fragment sizes for only a fraction of the BACs you would look at (that is an important point). I will also save some paper by pretending that the BACs are a little shorter than normal and produce fewer restriction fragments than normal.

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(i) From the data and background information provided does BAC-1 derive from the same region of the genome as (i.e. does it genuinely overlap) any of the four other BACs? Explain AND include some idea of how certain you are of your judgment.

(ii) If you wanted to start a map with BAC-1 and build a contig from there using all of the restriction fingerprinting data from the whole project (not just what I listed), how would you proceed?

(iii) If you used a smaller set of BACs where most had overlaps with other BACs but overlaps were generally small (say, less than 30% of BAC length) would restriction enzyme fingerprinting be successful in figuring out BAC overlaps? Explain.

(iv) If you were using the same (adequately large) set of BACs for restriction enzyme fingerprinting or for STS content mapping which map would be better?
(a) consider resolution (the ability to order the relative end-points of different BACs) and
(b) consider your ability to detect any clones that had undergone a small deletion event during cloning or amplification,
and describe any unknowns that you think relevant to your answer.
Both the extent of a BAC within a map and certainty that the BAC has no cloning artifacts are important in selecting BACs for a minimal tiling path.

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Hello Student,
I have included a lot of explanation in the answer, so, that it is easy for you to understand.

(i) If you do not know the sequence then it is hard to judge because a similar profile can arise in another BAC, although the chances are quiet low. Let us say, the sequence is similar, then it looks like that BAC1 is derived from the same region of the genome as any of the other BACs.

(ii) You would first compare the sizes of the products and use a program, which is desinged for automated size comparisons; then perform contig building to find overlaps in BACs representing the genome. This is method is extremely useful when you do not have prior knowledge regarding the grouping of the BACs. There is a method called shotgun approach. You can also digest the BAC with the a restriction enzyme to get fragments that are 1-2kb in length and then subclone into a vector and use the primers to amplify them and then sequence them. After that the overlaps between sequences are identified, and are assembled into contigs, and a consensus sequence is obtained.

(iii) A 30% overlap of a BAC of 200kb means that ...

Solution Summary

This job overviews restriction enzyme fingerprinting.

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