WSSP-04 Chapter 4 Mapping

Transcription

1 Chapter 4 Restriction Mapping a Clone Restriction enzymes are often used to move a fragment of DNA from one vector to another. This is commonly referred to as cloning a DNA fragment. Restriction enzymes are also used to analyze the clones, through a process that is called restriction mapping. This section will briefly describe how to map a clone. In order to clone a DNA fragment from one vector to another the DNA is first digested with one or more restriction enzymes. In the next step, the DNA fragment of interest is purified from the digest and then ligated into another vector that contains compatible ends. Sometimes the fragment is not purified and the whole mixture of fragments from the digestion is ligated into the new vector. The ligation mixture is then used to transform bacteria and cells are plated on selective media. Since bacteria can not be efficiently transformed with linear DNA, only those DNA molecules that are ligated into a circle and that contain an origin of replication and the selectable marker (such as ampicillin resistance) will be able to transform the bacteria. However in many cases not all of the transformants (clones) will contain the desired product. For example, if a mixture of fragments was used in the ligation, then it is possible that any of several fragments could be inserted into the vector. Alternatively, in some cases the vector may reclose without an insert. Since many different types of transformants are possible, it is often necessary to screen through the transformants to obtain the proper clone. This is usually done by preparing small amounts of several different transformants (minipreps) as described in the previous lecture, and then performing a test digest on the plasmids to screen for clones with the expected number and size of fragments. A RI PstI PstI A B RI PstI HindIII RI HindIII EcoRi/HindIII Digest A B Purify Frgs. A B Ligate PstI AB BA HindIII RI HindIII RI PstI HindIII RI PstI B HindIII Restriction mapping is also used to analyze a clone with an unknown insert. This is commonly done to get an idea what restriction sites are in the insert and how large it is. These sites can then be used to subclone smaller fragments of the insert into other vectors. In Lab 7 we will be performing this type of analysis on the DNA from random cdna clones that were prepared in Lab 4. You will also be doing this analysis on the clones you prepare this summer back at the school next fall. 4-1

2 a. Determining the size of the insert of a clone from the C. remanei cdna Library Generating a Restriction Map of the cdna Plasmid: The DNA sequences of virtually all cloning vectors have been determined. As a consequence, the recognition sites for all known restriction enzymes that are present in the vectors are known. The depiction of a group of restriction sites in a plasmid is called a restriction map (shown below). Restriction maps can be represented in a linear or circular fashion. For vector maps, an arbitrary site is chosen to be position 0 and then nucleotides are numbered sequentially. Thus, numbers represent the number of nucleotides away from the origin that a specific restriction site can be found. These numbers can be used to calculate the sizes of restriction fragments resulting from restriction enzyme digestions. Note the clustering of restriction sites in one region of the vector ptripleex. This region includes as many restriction sites as possible and is called a polylinker or a multiple cloning site (MCS). The polylinker region can be digested with an enzyme or a combination of enzymes to accommodate the insertion of a DNA fragment with complementary ends. C. remanei insert 4-2

3 1. Determining the size of the insert from the EcoRI digest. In recombinant DNA experiments it is not uncommon to isolate a gene of unknown sequence inserted into a vector of known sequence. This cloned DNA can then be easily purified and studied. The first step in working with a newly cloned sequence is to construct a restriction map. How is this done? The plasmid that was isolated from a library that was constructed by inserting random cdna fragments into the EcoRI site of ptripleex. (A map is shown on page 7-6.) The first step is to determine what size is the insert. The plasmid was cloned by adding EcoRI sites onto the ends of the cdna fragments. The ptriplex plasmid was then digested with EcoRI to generate a linear fragment. The EcoRI ends of the cdna were then ligated with EcoRI ends of the vector to produce the plasmid in the library. The plasmid should retain both of the EcoRI sites. Digesting the plasmid with EcoRI should therefore generate two fragments. One of the fragments will be the vector backbone. Since we know for the plasmid map that the vector is 3.6 Kb. One of the fragments should migrate between the 3.0 and 4.0 Kb markers on the gel. The other band on the gel corresponds to the cdna insert. Aligning the position of the fragment with the marker fragments will give you a rough estimate of the size. In the figure below the second fragment is migrating at approximately the same position as the 2.0 Kb marker. The insert is therefore 2.0 Kb in size. A sample of the uncut DNA is also shown on the gel. This is a useful control to show that you restriction digestion has worked. If it has not then your cut sample would look very similar to your uncut DNA. The uncut DNA will often run as three separate bands. The smallest is a supercoiled fragment which runs at approximately half the size of the plasmid (in this case about 2.8 Kb). If one strand of the DNA gets nicked it relaxes the supercoil an the DNA runs as a large open circle (12 Kb). This usually runs about twice the size of the plasmid. If both the strands break on the same location on the plasmid this will run as a linear (5.6 Kb). 4-3

4 2) Additional Mapping of the C. remanei clones: 1. The first step is to examine the vector for known restriction sites. Usually enzymes that are known to cut once in the vector are good choices for the first round of mapping of the cloned insert. Enzymes that cut infrequently recognize a sequence of six (1/4096 bp) or more nucleotides. Enzymes that cut many times, such as enzymes that recognize four base sequences (on average 1 digest per 256 bp), generate too many fragments in the vector and insert to be able to generate a map. Looking at the map of the plasmid on page 3-12 one can find a number of 6 bp cutter sites on either side of the insert (EcoRI site), such as SmaI and KpnI on one side and PstI, BamHI, XbaI, XhoI, and HindIII on the other side. Each of these are unique sites in the vector. It is sometimes useful to try enzymes that do not cut in the vector in case there are sites for these enzymes in the insert. 2. Digest the clone with individual enzymes which are known to have only one site in the vector. Digests are run out on an agarose gel and the size of resulting DNA fragments are determined. Three outcomes are possible: a. The DNA is not digested: The digest of the plasmid with ApaI (lane 5) looks identical to the uncut control (lane 1). It is possible the digest failed for a trivial reason (the enzyme was not added to the digest, it was denatured, or the digest was done at the improper conditions, etc). However, it is also possible that this site is not present on the plasmid. We know from inspection of the known sites in ptriplex that it is not present on the vector. Since it did not cut the clone we can conclude that it is also not present on the insert. b. The plasmid could be cut once, i.e., linearized. This implies that only the vector restriction site for that enzyme was recognized in other words, the insert does not appear to have a restriction site for that particular enzyme within it. In the example, the BamHI digest (lane 3) shows one fragment. This indicates that there are no BamHI sites in the insert and only one in the vector. The BglI digest (Lane 4) also shows one band. Since there are no BglI sites in the vector, ptripleex, what does this indicate? c. More than one fragment could be generated. This implies that there is a restriction site for that enzyme inside the uncharacterized insert fragment. ***For a circular plasmid, the number of total sites for a given plasmid equals the number of fragments generated.**** 4-4

5 For example, the EcoRI digest (lane 2) shows two fragments. Since the construct was made by inserting the cdna fragment with EcoRI ends into the EcoRI site of the vector ptriplex we would expect to generate two fragments (as in the example for determining the size of the insert). It also indicates there are no EcoRI sites in the insert, only sites on either end of the insert. This digest is useful to determine the size of the insert. In this example, the insert is approximately 2 kb (1 kb = 10 3 base pairs). This is useful information, since when adding up the size of the fragments in a digest they should always equal about 5.6 kb (3.6 kb of the vector and 2 kb for the insert). If the addition of the fragments does not equal 5.6 kb, then there may be a complication with the digest, such as two fragments being counted as one (referred to as a doublet) or an incomplete digest that produces too many bands. The HindIII digest (Lane 6) also has 2 fragments and therefore, in addition to the HindIII site in the vector, there is one site in the insert. In the next step we will determine where this site is. 3. Determine the positions of unknown restriction sites. If a digest produces two fragments and you know that one of the sites is in the vector, then it is possible to determine where the second restriction site is in the cloned insert. For example, the HindIII digest shown in lane 6 shows that the clone has two HindIII sites that generate fragments that are 1 kb and 4.6 kb. We know that the vector has one HindIII site on the right side of the EcoRI site. We can map the position of this site as follows: a. We know the relative position of the HindIII restriction site in the vector. b. In order to generate a fragment of 1 kb there must be an HindIII site 1 kb away from the first site. There are only two possibilities for the positioning of this restriction site (see the figure below). If the second site is was at the position shown in A then it would have to be in the vector sequence. Since we know that there is only one site in the vector and the sites do not spontaneously appear then we know that this possibility is not correct. Therefore the map shown in B with the site in the middle of the inert, 1 Kb away from the HindIII site in the vector is correct. 4. Double Digests: Enzymes that do not cut the vector can also be useful in mapping. For example, if you found that BglI (an enzyme known not to cut the vector) cleaved your DNA once, you could map that site. All you have to do is follow up on your observation by cutting the plasmid with two enzymes BglI and a second enzyme that you know cuts at a specific site in the polylinker of the vector, such as BamHI. Remember: When 4-5

6 performing double digests it is essential to determine if both enzymes can cut in the same buffer.the size of the doubly digested fragment tells you the position of the BglII site (Note that such an analysis would be complicated if you had a second site for BamHI in your insert!). In the example the BglI BamHI digest gives two fragments. On of the fragments is approximately 1.5 Kb indicating the two sites are 1.5 Kb apart. Since we know the location of the BamHI site in the vector and that BglI does not cut in the vector then that would indicate the BglI must digest in the insert and that it is 1.5 Kb away. The collected restriction digest data can be pulled together to produce a map: Other things to be aware of when analyzing restriction digests: 1. Uncut DNA. Solutions of undigested plasmid include a mix of DNAs of the same molecular weight but different topology. The three forms are superhelical circular, nicked circular and linear (some DNA gets broken). These forms also migrate toward a positively charged electrode and usually appear nearest to the well. They tend to migrate more slowly than digested DNA fragments, but their migration varies according to ethidium concentration, agarose concentration and buffer. The uncut forms of DNA can be quite confusing in restriction analysis. It is important to include a control sample of undigested DNA in your experiments to enable you to tell if your plasmid was digested or not. 2. Partial digestions: Sometimes the restriction digestion reaction does not proceed to completion. This partial digestion can occur for a number of reasons: if buffer conditions are not optimal, if DNA is digested for too short a time, if DNA is methylated, or if DNA is contaminated with protein or phenol. As you would expect, partial digestion can be very confusing when you are attempting to construct a restriction map. To recognize a partial digest first, the presence of bands that co-migrate with the uncut control suggests that cutting was inefficient (of course there may not be a restriction site there to begin with). Second, partials are characterized by faint bands that correspond to the size of two adjacent fragments in the plasmid. Note that because the ethidium-stained DNA fluoresces in proportion to molecular weight, large fragments should be brighter than smaller ones. The presence of faint large bands suggests a small amount of partial digestion product. Finally, if the size of the fragments is added up, then it will be more than the entire plasmid. 3. Size compression: As the fragments get bigger it is harder to resolve them on the gel. The percentage of agarose we use in gels in the lab are good for resolving fragments in the Kb range. It is therefore very hard to distinguish the sizes of larger fragments >5 Kb with very good 4-6

7 accuracy. One could use a lower percentage gel to better resolve these fragments or perform double digests to generate smaller fragments to map the plasmid. 4. Star activity: Restriction enzymes are proteins that require specific conditions to perform at optimum efficiency. If these conditions are not used then the enzyme may work slower or the digest may not go to completion. For some enzymes improper conditions can cause the protein to change its substrate specificity. When this occurs the enzyme will recognize and digest other DNA sequences that are not its normal site. Conditions of too much enzyme, glycerol, or high or low ph or salt can cause this activity. 5. Size standards. It is essential to run size standards along with restriction digests of your plasmids so that you can estimate the size of the fragments you observe. In the laboratory we will use the BRL 1Kb Plus size standards Lane 1 = Uncut Control Lane 2 = Linearized Lane 3 = Doublet Lane 4 = Partial digestion Lane 5 = Size compresssion Lane 6 = Uncut digest Lane 7 = Linear w start activity Lane 8 = Markers 4-7

8 Another Mapping Example: A 2 Kb EcoR1 fragment of DNA is cloned into the unique EcoR1 site of a plasmid vector. A student digests the resultant clone with the following restriction enzymes: BamHI, SalI, EcoRI, BamHISal I, BamHIEcoRI, SalIEcoRI. The pattern of restriction fragments seen on a gel is shown. Bam HI Sal I Bam HI Sal I Bam H1 Sal I 6 Kb 5 Kb 4 Kb 3 Kb 2 Kb 1 Kb What can we conclude from this gel? 1. The first and second lanes only show one fragment. This indicates that there is one BamHI site and one SalI site in the vector. However since we have no information about the vector it is not possible from these digests to tell where these sites are in the plasmid. However we can determine that the total size of the plasmid is roughly 5.0 Kb. 2. The EcoRI digest shows two fragments. This was expected since we know the plasmid was constructed by cloning a 2 Kb EcoRI fragment into the vector. The band migrating at 2.0 must be the insert. The other band migrating at 3.0 Kb must be the vector. We have now determined the size of the vector. We can now begin to generate the map shown to the right. Vector 3. The BamHI SalI double digest also give two fragments. This is expected since we know the plasmid contains one of each sites. We know that these sites are roughly 2.0 Kb away from each other. However, we can still not place these on the map 4. The EcoRI BamHI double digest gives three fragments. Again this is expected since we know there are two EcoRI sites and one BamHI. Comparing the double digest to the EcoRI digest we see the 2.0 Kb fragment is still present but the 3.0 EcoRI fragment corresponding to the vector is no longer present and has been replaced with a 2.5 and fragments. This indicates that the BamHI site is in the vector 2.0 Kb 2.0 Kb sequence and is away from one EcoRI sites. We Insert Insert then add the BamHI to the EcoRI EcoRI EcoRI EcoRI map. However note that we or can not distinguish between BamHI BamHI these possibilities. Vector 2.5 Kb EcoRI Vector 2.5 Kb 2.0 Kb Insert 3.0 Kb EcoRI 4-8

9 5. The EcoRI SalI double digest also gives three fragments. Again this is expected since we know there are two EcoRI sites and one SalI. This time comparing the double digest to the EcoRI digest we see the 3.0 Kb vector fragment is still present but the 2.0 EcoRI fragment corresponding to the insert is no longer present and has been replaced with a 1.5 and fragments. This indicates that the SalI site is in the vector sequence and is away from one EcoRI sites. We then add the SalI to the map. However note that again we can not distinguish between these four possibilities. SalI EcoRI BamHI SalI EcoRI 1.5 Kb Insert A Vector 2.5 Kb 1.5 Kb Insert C Vector 2.5 Kb 1.5 Kb Insert EcoRI EcoRI B BamHI Vector or EcoRI EcoRI BamHI 2.5 Kb 1.5 Kb Insert D Vector 2.5 Kb SalI EcoRI SalI EcoRI BamHI 6. Finally, if we go back to the BamHI SalI double digest we know that the two sites have to be 2.0 Kb away from each other. Looking at different possible maps we see that the BamHI and SalI sites are only 1.0 KB away in models A and D. We can therefore eliminate these two possibilities leaving B and C as possibilities. However note that again we can not distinguish between these possibilities because we do not know anything more about the vector or insert. If we found a map of the vector we could determine where the BamHI site is in respect to the rest of the vector and be able to choose one of the models.. 4-9

10 Name: School: Today s Assignment: 1. Draw a gel with markers, cut and uncut plasmid for a clone that contains a 3.0 Kb insert in ptriplex. 2. Draw a gel with markers, cut and uncut plasmid for a clone that contains a 3.0 Kb insert in ptriplex in which one of the EcoRI sites was destroyed during the ligation step of cloning the plasmid. 3. Draw a gel with markers, cut and uncut plasmid for a clone that contains a 3.0 Kb insert in ptriplex in which the EcoRI enzyme was not added to the mix. 4-10

11 Name: School: 4. A DNA fragment of unknown size is cloned into the Xba I site of the multiple cloning site (MCS) of a 5 Kb vector. A student digests the clone with the following combination of enzymes: BamHI, EcoRI, XbaI, BamHIXbaI, XbaIEcoRI, BamHIEcoRI. The pattern of restriction fragments seen on a gel is shown on the diagram below. Bam HI Xba I Bam HI Xba I Xba I Bam HI 11 Kb 9 Kb 6 Kb 5 Kb 4 Kb 3 Kb 2 Kb 1 Kb What is the size of the DNA insert? Draw a circular map of the clone. 4-11

12 Name: School: 5. A DNA fragment of unknown size is cloned into the XbaI site of a 3 Kb vector that does not necessarily contain a MCS. A student digests the clone with the following combination of enzymes: BamHI, EcoRI, XbaI, BamHIXbaI, XbaIEcoRI, BamHIEcoRI. The pattern of restriction fragments seen on a gel is shown on the diagram below. Bam HI Xba I Bam HI Xba I Xba I Bam HI 8 Kb 6 Kb 5 Kb 4 Kb 3 Kb 2 Kb 1 Kb a) What is the size of the insert DNA? b) Draw a circular map of the clone. 4-12

13 Advanced Restriction Problems 1. What three types of ends can be generated after restriction digestion of DNA? 2. a) Using the chart provided on page 226 of the NEB manual, which buffer would you use to set up a digest with: MspI NotI AflII BamHI AluI ApaI AhdI AatII / AlwI AciI / AvaI MluI PmlI SmaI EcoRI MspI and MluI: NotI/PmlI AflII/SmaI BamHI/EcoRI b) A student sets up an AflI/SmaI digest in the appropriate buffer and a 2-fold excess of enzyme and incubates the digest at 37 C. He finds that even after 3 hrs he is only getting partial digestion of the plasmid. Why? What would you do to solve the problem? c) Another student sets up a Tth111I digest in buffer #4 and incubates it at 37 o C. After a one hour digestion she finds it did not cut completely. What is the most likely problem? 3. Polylinker sequences in vectors contain multiple restriction sites that are closely spaced together. These sites are often very useful for cloning DNA fragments that have ends generated by two different restriction enzymes. However, sometimes the close distances between sites can be a problem. Some restriction enzymes are inefficient in trying to cut a site that is near the end of a fragment. On page 238 of the NEB catalog a guide is provided that describes the efficiency of cleavage by some of the popular enzymes. a) A graduate student wants to digest puc19 with BamHI and SmaI. (A map of puc19 is on page 2 of the notes for Lecture 2 or page 216 of the NEB catalog.) Should she digest the DNA with both enzymes at once? If not, which one should she use first? b) How should she set up a digest of puc19 with KpnI and EcoRI? 4. A student wants to prepare a restriction digest of 3 mg of plasmid DNA with 80 Units of BamHI in a total volume of 50 ml. How much of his DNA stock (at 500 mg/ml), enzyme (20 U/ml), 10X buffer and water should she add to the tube? DNA (500 mg/ml) BamHI (20 U/ml) Buffer #1 (10X) Water 4-13

14 5. A student is thinking about an upcoming exam and is not concentrating on his work. In setting up some restriction digests he makes the following mistakes: a) He adds 5 ul instead of 0.5 ul of EcoRI (20 Units/ul) enzyme to a 20 ul reaction. b) He adds 10 ul of the 10X stock of buffer #2 to a 50 ul KasI digest. c) He adds 5 ul of buffer #2 to a 50 ul BglII digest. Which of these mistakes might be the most problematic? If he catches his mistakes, which of these digests should he set up again? 6. Several groups in a laboratory course are going to digest two plasmids that were prepared from two different strains. One group digests the plasmids with Sau3A, while the other group uses MboI. One of the plasmids that was supplied to all of the groups was puc19 that was prepared from strain XYZ1. When the students compare their digests, the band pattern from each group is identical. Why? How many fragments would be expected from a digest of puc19 with Sau3A? Or with MboI? The students then compare Sau3A and MboI digests of pbr322 that was prepared from strain XYZ2 and supplied to all of the groups. The group that digested the DNA with Sau3A saw a large number of fragments while the other group that cut with MboI saw only uncut DNA. The students figured they messed up the reaction and so they set up a new digest. However, they got the same result. Why? The students go back and check the two strains that the plasmids were prepared from. The only difference between the two strains was that one was dam and the other dam-. Does this make sense? Can you tell which plasmid was prepared from which strain? 7. Below is an abbreviated map of pnb8 and a diagram representing the results of a restriction analysis of a cdna clone from a library that was made by inserting fragments into the StyI site of the vector. a. Construct a map of the this new clone b. What size fragments would be expected for a StuI/EagI digest? A SalI/BamHI? 4-14

15 c. Assuming there are no PvuII sites in the insert, what would be the number and size of the fragments from a PvuII digest? 8. A large portion of molecular biology involves cloning; i.e. moving fragments of DNA from one vector to another. Restriction enzymes are not only used to obtain specific fragments, but they are often used to screen for the proper products. A student wishes to subclone a portion of a large genomic clone, ptk2, into puc19 for further analysis. The genomic clone contains a 6 KB insert into the BamHI site of the vector YEp24 (p. 222 of the NEB catalog). A partial restriction map for the genomic clone is shown below. The student decides to digest the genomic clone with EcoRI and XhoI, and although she tries to gel purify the 2.5 Kb fragment (bp ) she feels that it is likely that she also got some of the other fragments as well. She digests puc19 with EcoRI and SalI and ligates the digested vector with the purified fragment. After she transforms the ligation into bacteria she selects the transformants on LBampicillin media. She does double stranded minipreps on several colonies and screens for the proper construct by performing single digests with PvuII and then HindIII. 4-15

16 a) How many bands should she expect of the EcoRI/XhoI digest of ptk2? What are the sizes of these bands? b) How was she able to clone the insert if the fragment had EcoRI/XhoI ends while the vector had EcoRI/SalI ends (hint: check restriction sites for XhoI and SalI)? Does the desired clone contain any EcoRI, XhoI, or SalI sites? c) She runs her PvuII digest on the gel and gets the patterns for the clones shown below: What does this gel tell her? What is the likely explanation for lane #4? d) She next runs the HindIII digest and gets a gel that looks like the one shown below. Does this help her distinguish which clone(s) are the correct ones? What is the most likely explanation for each of the clones? 9. A student wants to insert a DNA fragment into the Tth111I site of a vector. She consults the NEB catalogue on the enzyme (see below) and sets up a digest of 10 mg of plasmid DNA in a 100 ml reaction using the NEB buffer #4. She digests the DNA using 20 Units (from a 20 U/ml stock) of enzyme and incubates the reaction at 37 o C for 2 hours before doing her cloning. In screening her clones, she finds that most them are the parent vector. She goes back and examines her digest on a gel and finds that most of her parent vector DNA was uncut. What is the most likely explanation for her results? 10. Why are markers always run on the gel? 11. Using the NEB buffer chart provided, which NEB buffer (1, 2, 3 or 4) would you use to set up the following restriction digests? 12. The two most important rules in working with enzymes are: 4-16

17 13. A 4.5 kb genomic fragment is cloned into the HindIII site of the pks vector to make pxy200. A partial map of the pks vector is shown with all of the unique restriction sites in the polylinker. pks (3.0) kb) XbaI BamHI EcoRI HindIII XhoI A series of restriction digest is performed on pxy200 to map the insert. A gel of the digest is shown below. Assume that all digests are complete. Determine a map of the plasmid. (Lane 2-HindIII, 3-EcoRI, 4-BamHI, 5-XbaI, 6-XhoI, 7-EcoRI/BamHI, 8-EcoRI/XhoI, 9-EcoRI/XbaI, lanes 1 and 10-markers) 4-17