|
Table of Contents
|
Purpose
To determine if phenylthiocarbamide (PTC) haplotypes are observed in our population sample (Biology 205 students from 2012) with the same frequencies seen in other population samples.
Where we are starting from
This project is a continuation of a project we started in Biology 205 (Winter 2013). In 205 we attempted to determine each student's PTC diplotype by sequencing their PTC gene. Each student's DNA was isolated and their two PTC alleles amplified in a single reaction using PCR.
We were not able to determine the actual haplotypes for some students because we were sequencing both alleles simultaneously. This resulted in anyone who was heterozygous at two or more nucleotide positions giving inconclusive results.
To fully understand our previous results, you will need to familiarize yourself with the PTC background information. For now, you need to understand is that in order to identify the actual haplotypes of the students who were heterozygous at two or three positions, we need to sequence their PTC alleles separately. In order to sequence the alleles separately we will first need to make separate clones of each alleles using a technique called shotgun clone A cloning method where a mixture of more than one distinct DNA fragment is combined with a vector. During the ligation reaction, thousands of individual ligations take place and each time only a single fragment will be ligated to a single vector. Since there is no way to control which fragment will actually be ligated to the vector each time a ligation occurs, we can only distinguish the various recombinants after the ligation step is complete..
PTC background information is here.
The basic steps involved in producing recombinant DNA are outlined on the General DNA Cloning page.
Step I. Planning the cloning strategy
In order to plan a cloning strategy, you must first be familiar with the many available approaches and several molecular protocols. Since you are new to molecular biology the cloning strategy has already been laid out for you. However, by the end of the semester, you will be expected to be able to plan out your own hypothetical cloning strategy. The main things you need to decide before you start are
- how to obtain the DNA fragment(s) that will be cloned
- what vector to use
- what bacterial strain to use
- what screening strategy will be used determine to determine which bacteria carry the desired recombinant plasmid
Each group will start with one genomic DNA sample that was taken from one person in biology 205. You will not know the identity of the person; in fact this information was not tracked so even Lauri does not know. We will use PCR to obtain multiple copies of each allele (2 alleles per sample). Each group will then shotgun clone A cloning method where a mixture of more than one distinct DNA fragment is combined with a vector. During the ligation reaction, thousands of individual ligations take place and each time only a single fragment will be ligated to a single vector. Since there is no way to control which fragment will actually be ligated to the vector each time a ligation occurs, we can only distinguish the various recombinants after the ligation step is complete. the 2 alleles and determine the allele sequences.
Step II. Producing the starting material
The method used to producing starting material depends on what is available.
We will use PCR to produce the DNA fragment we wish to clone because this is the easiest way to fish out a small piece of DNA (~ 1000 bases) from the full human genome( ~ 3.16 * 109 bases).
We will clone the PCR products using a strategy called TA cloning A method for cloning PCR products that takes advantage of Taq polymerases tendency to add a single overhanging 3'-dA to the end of PCR products. The PCR products are ligated to a linear vector that has complimentary overhanging 3'-Ts. The two overhangs stick (H-bond) to each other, allowing ligase to easily ligate the PCR product to the vector . In order to TA clone, we need specially treated vector DNA that has already been digested and "tailed" with a few Ts. This vector is tricky to produce so we are going to use a commercially prepared product call pGEM-T® easy, purchased from the company Promega.
Step III. Prepare the DNA fragments for cloning
The vector must be linearized so that the DNA fragment you want to clone can be inserted into the vector. Usually this is done using restriction enzymes.
For our project we are using commercially prepared vector that was linearized using an enzyme that produced blunt ends. The 3' ends of the linearized vector were then modified by the addition of a single thymidine. See Promega's site for more information on how the vector was prepared.
The PCR products will have 3'-As added by Taq polymerase, in a template independent manner, during the amplification process (this is a quirk of the Taq enzyme that we take advantage of). We will need to quantify the PCR product before we can determine the amount to add to the ligation reaction.
Step IV. Ligation
In order to create a functional recombinant plasmid, we need to covalently join the vector and a PCR product together using the enzyme DNA ligase. As with all enzymes we use, ligase is naturally occurring. In vivo, DNA ligase is used during DNA synthesis to join DNA ends in lagging-strand synthesis of DNA. The DNA ligases commonly used in cloning originated from either bacteria or from bacteriophages.
DNA ligase catalyzes the formation of phosphodiester bonds between adjacent 3'–hydroxyl and 5'–phosphoryl termini of nucleic acids. To bring the 3' and 5' ends of two DNA molecules together, it is helpful to have complementary bases on the two molecules (called 'sticky-ends'). In the case of TA cloning, the 3'-T overhang added to the vector will base-pair with the 3'-A that was added to the PCR products during amplification.
We are actually cloning two distinct DNA fragments (we will call them ProductOne and ProductTwo for simplicity) because we are using DNA extracted from individuals who are heterozygous for the PTC allele. This type of cloning is called shotgun cloning because we have no control over which fragment gets inserted into the vector each time a ligation reaction occurs.
Step V. Screening the newly constructed plasmids
Ligation reactions often produce more than one product and screening is required to find the desired recombinant. Screening involves three main steps: transforming bacterial cells with the ligation products, selecting for bacteria carrying recombinant plasmid, and checking the plasmids to make sure they carry the correct insert.
Transformation
Transformation refers to the process whereby cells uptake 'naked' foreign DNA. When cells are amenable to transformation they are referred to as competent. Occasionally, transformation occurs naturally, but most bacterial species need to be made competent by chemical or mechanical treatments. We will use the traditional calcium-chloride method to produce competent cells.
Screening transformants
Antibiotic selection
During transformation only a subset of the bacteria will actually take up DNA. In order to identify which bacteria are successful transformed, all cloning vectors have a selection marker. Often this marker is a gene encoding antibiotic resistance. Following transformation, the bacteria are plated on the appropriate selective medium (the antibiotic in the media must match the resistance gene encoded by the plasmid). Only bacteria that are transformed with the vector will be able to grow on this media. Our vector, pGEM-T easy, carries the gene for ampicillin resistance (β-lactamase).
Blue/white screening
Depending on the vector chosen, we may also be able to screen for bacteria that carry a recombinant vector (vector containing an insert in the MCR). In our case, visual screening is possible because pGEM-T® easy carries a lacZ gene fragment. Bacteria carrying an insert in the pGEM-T® easy MCR will produce white colonies when grown on media containing X-gal. If no insert is present, the colonies will be blue.
Please see Blue/White Screening for a detailed explanation.
Identifying recombinant DNA
Once you have identified the colonies likely to carry the desired recombinant, the recombinant plasmids harboured by the colonies must be directly screened. Even when you expect only 1 recombinant type, it is important to confirm the correct construct is present, as visual screening methods are not 100% reliable. You would hate to find out several experiments down the road that you had not constructed the desired clone.
There are several approaches to screening the recombinant plasmids; we will use restriction mapping. Because we know the sequences of both the vector and the insert, we can predict the sizes of fragments that will be produced when we digest the desired recombinant. It is simply a mater of isolating the plasmids and digesting them with restriction enzyme(s) that will produce a diagnostic set of fragments (visualized using agarose gel electrophoresis).
Isolating the recombinant plasmids
To purify plasmid DNA, we will use a kit manufactured by Fermentas. This kit uses the tried and true method of alkaline lysis followed by neutralization to lyse the bacterial cells and separate the plasmid DNA from most other cell contents. The plasmid is further purified using a silica membrane. The ultra clean plasmid DNA produced using this kit will be compatible with all downstream cloning steps.
See Isolating plasmid DNA for more informations.
A molecular biology kit is a commercially prepared set of reagents for a specific protocol. Generally some of the reagents and/or the protocol is proprietary in nature and has been extensively developed and tested to make the protocol both simpler and more dependable than the ‘from scratch’ version.
Restriction mapping the recombinant plasmids
Finally, purified plasmid DNA is digested with restriction enzymes in order to produce diagnostic size fragments. The fragment sizes are then determined using agarose gel electrophoresis. Restriction enzymes are chosen by referring to the plasmid and insert sequences. For more on how restriction enzymes work, and how to choose the appropriate enzymes see here.
Step VI. Using the recombinants to answer our question
Once we have confirmed that we have the correct constructs, we can sequence the purified plasmids. We will send our plasmids to Eurofins MWG Operon for sequencing.
Once we have the sequences in hand we can finally directly address our initial purpose!
Remember, each individual sample had two distinct products PCR products that were identically sized. We will not know which colonies harbored which alleles until we have the sequencing results.