Question

1-After PCR, what types of procedures are done with the amplified fragments? 9. What adjustments in the PCR process might you do if you want to clone the PCR product?

2-What adjustments in the PCR process might you do if you want to clone the PCR product?

Answer 1

SOLUTIONS:

The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours. While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR is commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment. However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described. This chapter provides an overview of different types of PCR methods, applications and optimization.

Amplification and quantification of DNA

Fig 1 : Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar and the body of English king Richard III.

Quantitative PCR or Real Time PCR (qPCR, not to be confused with RT-PCR) methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method consists of using fluorescent dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA takes place. An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique, called RT-qPCR, allows for the quantification of a small quantity of RNA. Through this combined technique, mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of PCR,[24] increasing chances for detection of genes associated with genetic diseases such as cancer.Laboratories use RT-qPCR for the purpose of sensitively measuring gene regulation.

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.

Cloning and Sequencing your PCR Product

I. Cloning

Use the Invitrogen TOPO TA Cloning kit. Please read the instruction manual carefully before starting. Invitrogen has changed the kit several times in the last year. The latest manuals are versions J and K as of 9/00. Please note that the kits no longer contain b-mercaptoethanol or stop solution but now contain a salt solution which is added to the cloning reaction.

The TOPO vector has an overhanging 3' T. Your PCR product must have an overhanging 3' A. Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need longer extension times.

It is a good idea to purify your PCR product on a Qiagen PCR purification column before cloning to get rid of your PCR primers. Otherwise you may get a lot of clones that only have your primers as inserts.

The cloning kit has 2 parts: 1) A box containing the vector and salt solution which is stored at -20C and can be found in the enzyme freezer. 2) A box containing the competent cells, SOC, and pUC18 control DNA that is stored at -80C and is in the bottom compartment of the freezer next to the gel room. Handle the competent cells with care. Thaw them on ice and only thaw them when you are ready to use them. If they warm up they will not transform. The SOC is very rich medium. Be careful not to contaminate it or you will contaminate everyone else's transformation. Do not use it if it is cloudy or has anything growing in it. Return it to the freezer as soon as possible.

Before you start the transformation you need to do several things: 1) Warm a waterbath or heat block to 42 degrees for the heat shock step. 2) Thaw the SOC and let it come to room temperature. It's best to thaw it on your bench. It's more likely to get contaminated or knocked over if you put it in a waterbath. 3) Make sure you have X-gal and Amp plates. The X-gal is in the sequencing box on the top shelf of the -20 freezer at the end of the lab. It is 50mg/ml in N,N-dimethylformamide. Amp plates are made by the kitchen and are in the coldroom at the end of the lab. You may want 2 plates for each transformation. To ensure a good density of colonies you can plate 50ul on one plate and 200ul on the other. Dry the plates in the 37C incubator with the lids ajar for at least 15 minutes. Spread 40ul of X-gal on each plate and keep at 37C for at least 30 minutes. Leave them there until you are ready to plate.

The new manuals have some very vague instructions so here are some suggestions: 1) For the cloning reaction the instructions say to add 0.5-4ul of PCR product, but no concentration is given. Most people use a total of 20ng. Incubate at room temperature for 5-10 minutes. 2) For the transformation, the instructions say to incubate on ice for 5-30 minutes. Twenty minutes works well. 3) After adding the SOC, the instructions say to incubate at 37 degrees for one hour. People have found that 30 minutes is sufficient. Incubation is done in the roller drum in the 37 degree incubator.

The TOPO kit contains a very useful quick reference card to use while you are transforming. A card is taped to the front of the enzyme freezer. Please return it when you are through. You should do 2 controls:1) vector alone. It should give you very few colonies. The vector should not be able to self-ligate. 2) pUC18 to check transformation efficiency. See instructions on page 10 of the manual.

After plating, incubate the plates overnight at 37 degrees. Remove them the next morning and store at 4C until ready to pick colonies.

II. Preparing DNA

There are 2 ways to prepare your DNA for sequencing: minipreps and PCR. The PCR method is faster and easier but occasionally people have found that it does not work well on their construct. Whatever method you choose, if you've never done this before, I suggest you do a few samples and send them for sequencing to see if you've got the method working before doing 100 at $7-10 a sequence. Also we have a Qiagen vacuum manifold (on Pam's bench) that can be used for both methods if you have a large number of samples. It uses strips of 8 column filters and processes 48 samples at a time, so it is best to do your samples in multiples of 8.

PCR method

Decide how many sequences you want to do. You will need to pick 50 to 100% more colonies than you need because some will not work and some will be the wrong size.

1) Make up the appropriate amount of 5xPCR master mix. You will need 20ul for each clone.

Stock Resultant

PCR Buffer 300ul 10x 5x

JP21.145 F primer 60ul 100uM 10uM

JP21.144 R primer 60ul 100uM 10uM

dNTPs 120ul 5mM(ea.) 1mM(ea.)

Water 54ul

Taq 6ul Non-commercial

Total 600ul

2) Fill enough 0.2ml strip-tubes or PCR plates with 80ul of water to accommodate all your colonies. Pick a white colony with a 10ul pipet tip. Place the tip on the end of your P10 pipettor and expel the colony into 80ul of water by pipeting up and down 5 times. Heat in PCR machine on INSTANT INCUBATE at 95C for 5 minutes to lyse the cells.

3) Add 20ul of master mix to each tube. Pre-heat to 94C for 4 minutes, then do 20 cycles of (92C for 1 min, 50C for 1 min, 72C for 1 min).

4) To check the quantity and quality of your DNA, run 3ul on a 2% agarose gel along with 4ul of Gibco low DNA Mass ladder. It is not necessary to run all your samples. A few of each type will do.

5) If you just have a few samples, purify using the Qiagen Qiaquick PCR purification kit and follow the directions in the handbook.

If you have a large number of samples, use the Qiaquick-8 PCR purification kit and the vacuum manifold. It's best to have someone help you the first time you use the manifold. However, a few notes: 1) Apply,turn off, and release the vacuum slowly; otherwise your samples will spray everywhere and cause cross-contamination.

2) To elute your DNA, place the plexiglass block in the bottom of the manifold. Place an ISC 96-well plate on the block and the top of the manifold holding the columns over the plate. Make sure the columns and the wells are aligned and that the tip of the column is below the top of the well. Add 80ul of elution buffer to the center of the column. Let stand for 5 minutes and then turn on vacuum for 5 minutes. Cover with a microseal film for storage.

6) Run 3ul of all your samples on a 2% agarose gel and quantitate.

Minipreps

Pick a single white colony into 4ml of luria/amp. Ampicillin (50mg/ml) is in the sequencing box. Make a 1 to 1000 dilution in luria broth for a final concentration of 50ug/ml. Use either 14 ml round-bottom Falcon tubes (cat#2059) or 15ml conical centrifuge tubes with the blue tops. You must poke holes in the tops of the conical tubes for aeration. Grow overnight in the 37C incubator roller-drum for 12-14 hours. If you grow them longer the Amp may go off.

Spin down cells at 3000 rpm for 10 minutes in the Beckman Allegra tabletop centrifuge. (NOTE: If you are not processing them immediately, keep the cells at 4C and spin them down just before processing. If it's going to be longer than a few hours before you process them, it's best to spin down the cells and freeze the pellets). After spinning, aspirate off the supernatant or carefully pour off and blot with a kimwipe.

There are 2 ways to process your minipreps. Use the Qiaprep spin miniprep kit for small numbers of samples. Follow the directions in the handbook.

For a large number of samples, use the Qiaprep 8 Turbo miniprep kit with the manifold. It's best to have someone show you how to use the manifold the first time. Follow the directions in the handbook. There are 12 steps in the protocol and below you will find notes to go with many of the steps:

Steps 1-3: Add buffers P1, P2 and N3 using an eppendorf repeat pipettor and the appropriate combitip. None of the pipettor setting/ combitip combinations allow you to add the 350ul of N3. You will have to add 250ul + 100ul.

Step 1: The P1 buffer stored at 4C contains RNase. Be careful not to slop it around the lab. Resuspend the pellet in the hood at the center of the lab. You will find a white microcentrifuge tube rack in the hood. The easiest way to resuspend the pellet is to run the bottom of the tube along the rack until no clumps are visible in the solution.

Step 2: P2 buffer contains NaOH. Do not wait much longer than 5 minutes before adding the N3 (neutralizing) buffer or you may irreversibly denature your DNA.

Using the manifold: 1) To set up the manifold, you need to use 2 different sets of filter strips at the same time. The turbofilters, (clear with a dark blue stripe) clear the lysates and go in the slots in the lid of the manifold. Fill any unused slots with plexiglass blanks stored on the bottom of the manifold. The qiaprep strips (light blue) bind the DNA and go in the plexiglass strip holder which is placed in the base of the manifold. Place the lid on the base. THE TURBO STRIPS SHOULD BE DIRECTLY ABOVE THE QIAPREP STRIPS. MAKE SURE THEY ARE PROPERLY ALIGNED OR YOU WILL LOSE YOUR SAMPLE. Unfortunately

the lid can go on in 2 orientations and one will cause misalignment. I have placed black X's on what should be the left-hand side of both the stripholder and the lid. 2) Apply, turn off, and release the vacuum slowly; otherwise you will spray your samples all over the manifold and cause cross-contamination.

Step 4: Maximum vacuum should not be used for this step. Use the vacuum line on Pam's bench with the vacuum regulator. Adjust the regulator to 200mbars before applying vacuum to the manifold. If all the lysate hasn't gone through the filters after several minutes and most of the flowthrough has stopped, slowly adjust the regulator to 400mbars. When the flowthrough has stopped and/or the samples are starting to foam, slowly turn off the vacuum. Any lysate that hasn't gone through by now isn't going to.

Step 5: Re-adjust the regulator to 200mbars before passing the lysate through the qiaprep column.

Steps 6-12: Maximum vacuum is used for the rest of the steps. Use the Matrix electronic multi-channel pipettor and Matrix tips (Pam's bench) to add the rest of the buffers.

Step 10: You need to get rid of all the PE buffer before proceeding. Blot the nozzles of the strips with a large kimwipe. If you've spilled any buffer around the tops of the columns, carefully open the lid and wipe it away. Put several layers of large kimwipes on the bench and rap the lid sharply until no more droplets appear. Change the kimwipes when necessary.

Step 11: Ignore directions in 11a and 11b. To elute your DNA, place the plexiglass block in the bottom of the manifold. Place an ISC 96-well plate on the block and the top of the manifold holding the columns over the plate. Make sure the columns and the wells are aligned and that the tip of the column is below the top of the well.

Step 12: Add 100ul EB buffer. Let stand 5 minutes before applying vacuum. After eluting sample cover with microseal film for storage.

Run 1ul of all you samples on a 1% agarose gel along with 4ul of Gibco low DNA Mass ladder. Quantitate your DNA.

III. Sequencing

There are several facilities you can use. I will list them in order of preference.

1) The Biopolymer Facility at Harvard. Web address: BPF.med.harvard.edu Cost: $10/sample. Advantages: very good quality of sequence, courier service picks up and delivers samples (Call Doug at 257-5754), has standard primers that they provide at no extra cost. Disadvantages: Not high-throughput. They think 30 samples are a lot. However, not really much slower than the other places.

Amounts of DNA and primer required: They call primers they provide " standard"

and ones you provide "custom". For some reason they ask for different amounts of template to use with the 2 types of primers.

Template Amount Primer Amount Total Volume

plasmid 550ng standard 0 12

plasmid 500ng custom 4pmol** 15

PCR prod. 13.6ng* standard 0 12

PCR prod. 12.5ng* custom 4pmol** 15

(*ng/100bp of PCR product; **3ul of our 10ng/ul primer stock)

2) MIT-HHMI-CCR Biopolymers Laboratory, Web address: Biopolymers.mit.edu

Cost: $13/sample. Advantages: Good quality sequences, reasonably fast turn-around time. However, they don't do runs on Friday. Disadvantages: Samples have to be delivered to MIT, the names they give to sequences are so long they are difficult to read and work with.

Amounts of DNA and primer required: For plasmids, 500-600 ng of DNA and 3.2pm of primer (2.5ul of our 10ng/ul stock) in a total of 12 ul. For PCR products, 10-20ng/100bp of PCR product and 3.2pm of primer in a total of 12ul. Less DNA is required as the size of your PCR product increases. Check the web site for details.

3) MGH Dept. of Molecular Biology DNA Core Facility, Web address: xanadu.mgh.harvard.edu. Cost: $7/sample. Advantages: They're close. You leave your samples in the refrigerator by the tearoom. High-throughput. They have a new ABI3700 96-sample capillary sequencer. Disadvantages: No quality control, no feedback. Sequence quality varies from fair to poor. If you decide to use this facility, sequence in both directions and check your sequences very carefully.

Amounts of DNA and primer required: For plasmids, 300-400ng DNA and 2.5ul of our 10ng/ul primer stocks in a total of 12ul. For PCR products, 20ng DNA/100bp of PCR product and 2.5ul of our 10ng/ul primer stock in a total of 12ul.

4) Dana Farber/HCC High-throughput DNA Sequencing Facility. This facility also has a new ABI3700 96-sample capillary sequencer. Their plan is to make the minipreps for you using Qiagen kits and then sequence the DNA. They will provide forward and reverse M13 primers. A great idea-unfortunately it didn't work very well. We tried it once and it took one month to get sequences that were so full of N's they were not useable. I wouldn't recommend using this facility at this time but if they get it running properly it could be very useful.