Basic Principles of Biotechnology

Objectives

By the end of this lesson you should be able to:

1. Describe how DNA is extracted and purified
2. Explain the basic principles of PCR amplification, restriction mapping, blotting, Sanger sequencing, and DNA cloning and screening 

This week we will learn the basic principles of biotechnology. In later special topic classes we will see how biotechnology is applied in the real world, including the bioengineering of food, identification of criminals, and finding cure for disease.

DNA Extraction and Purification

Often, the DNA that is to be studied must be first extracted and purified. This requires mechanical and chemical breakdown of cells to expose the DNA, followed by removal of unwanted molecules such as RNA and proteins. The illustration below shows a typical procedure for DNA extraction and purification.

After extraction and purification, the DNA may be digested with enzymes and may be cloned and stored into DNA libraries. It may also be amplified by a method called polymerase chain reaction (PCR) and further studied using restriction mapping, blotting, sequencing and other biotechnology methods. 

DNA Digestion

DNA is digested with enzymes called restriction endonucleases, or simply, restriction enzymes. Restriction enzymes are enzymes produced by bacteria as a defense mechanism to protect them from phage attack. They recognize and bind to DNA at a specific nucleotide sequence called a restriction site or recognition sequence. The enzyme then cut the sequence by cleaving the phosphodiester backbone of the DNA. The cuts result in DNA fragmentation. The fragment sizes vary due to the random distribution of restriction sites.

Recognition sequences possess a type of symmetry called a palindrome, where the sequences read the same from the 5’ to 3’ direction. This is similar to how some words read the same backwards and forwards, e.g. racecar, refer, madam, and kayak. The most common recognition sequences are 4-5 nucleotide sequences long.

Some restriction enzymes produce fragments with overhanging ends called sticky ends while others produce blunt-end fragments. DNA from a vector that is cut with the same restriction enzyme used to cut a host DNA, can stick to the sticky end of the host DNA. DNA ligase can then be used to covalently seal the phosphodiester backbone, joining them to produce a recombinant DNA.

The most commonly used DNA vector is bacteria plasmid. Suitable plasmids must meet the following criteria:

1. Contain several restriction sites that allow insertion of DNA fragment of interest
2. Must be able to replicate in host
3. Must have a marker gene to distinguish between cells that have taken up the vector from those that have not, e.g. gene that produces light or color, bacteria resistance, or ability to metabolize a certain substrate

The following figure illustrates how plasmid DNA can be incorporated into bacteria.

A limitation of plasmids is that they are small, and therefore can only take up small pieces of DNA. To address this problem, other vectors have been developed e.g.

1. Modified bacteriophage lambda
2. Bacterial artificial chromosomes (BACs)
3. Yeast artificial chromosomes (YACs)

DNA Libraries

The cloning procedure described above, starts with a mixture of fragments from the entire DNA (including introns and exons). This is called the “shotgun approach”. It is so called because no specific gene is targeted for cloning. Instead, many different recombinant plasmids are produced. The complete set of clones each carrying a part of the DNA of interest is called the genomic library. Scientists can access these libraries from a commercial source or sequencing center. The clones are stored in multi-welled plastic plates with one clone per plate.

If researchers are just interested in studying the proteins coded by the DNA, a complementary DNA (cDNA) library is needed. This library is constructed by first extracting the mRNA from the cells where the gene is expressed, and then applying reverse transcriptase to make a single-stranded DNA. The mRNA is then degraded by enzymes and a second DNA strand complementary to the first is made by DNA polymerase. The resulting DNA is called cDNA. Since the cDNA is made from many mRNA molecules expressed by many different cells, it is called a cDNA library.

Screening

Specific genes of interest can be identified by screening the library. However, you will need to start by knowing at least part of the sequence of the gene you are interested in. This may be done by first deciphering the amino acid sequence of the protein it codes for, or by knowing the sequence of a closely related specie. Once you know the sequence, you can create a complementary sequence, called a probe, and label it with a radioactive isotope or fluorescent tag.

To carry out the screen, the cells from each well is transferred to a defined location on a membrane made of nylon or nitrocellulose. The membrane is then treated to break down the cells and denature the DNA. The membrane is incubated in a solution containing the probe, resulting in hybridization of probe with single stranded DNA. To visualize the gene of interest, the membrane is placed under a photographic film, allowing radioactive areas to be exposed. The researcher can then go back to the original plate to pick out the colony that hybridized with the probe, and grow those cells for further studies.  

Polymerase Chain Reaction (PCR)

Cloning DNA using vectors and host cells is very labor-intensive and time-consuming. Thanks to the development of polymerase chain reaction (PCR), we have a faster way to replicate target DNA sequences. In this procedure, the DNA to be amplified is placed in a tube containing thermostable DNA polymerase I (Taq polymerase), Mg 2+ (cofactor), and the four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP – substrates for DNA polymerase).

In order to run PCR, you will need to know part of the target sequence. This is necessary to build the two primers that will attach to the opposite strands of the DNA template. A complete PCR cycle consists of the following steps:

1. Denaturation: Separation of DNA to single strands
2. Hybridization/Annealing: Attachment of DNA primers
3. Extension: Copying of DNA by DNA polymerase

The number of DNA strands double in each cycle. Each cycle takes 2-5 minutes. Hence, in 30 cycles, a single molecule is amplified to more than a billion copies.

A type of PCR called reverse transcriptase PCR (RT-PCR) create copies of cDNA starting from mRNA. Another type called Quantitative Real-Time PCR (qPCR), allows the researcher to determine the amount of PCR product in real time.

Restriction Mapping by Gel Electrophoresis

Following PCR amplification, the DNA may be cut with restriction enzymes and then placed in wells of an agarose or polyacrylamide gel for molecular weight separation. An electrical charge is applied to the gel, allowing current to flow from a negatively charged end (anode) to a positively charged end (cathode). Since DNA is negatively charged, it moves toward the cathode. The speed at which it moves depend on its molecular weight, where the smaller fragments move the fastest. At the end of the run, you get bands of varying molecular weight fragments which can act as a finger print, or map of the DNA.

Blotting

Following fingerprinting, DNA can be further studied by blotting. The first of these techniques to be developed was Southern blotting. In this technique, the DNA on the gel is denatured to single strands and then transferred to (or blotted onto) a nylon or nitrocellulose membrane. A DNA probe complementary to the sequence of interest is then added to the membrane to allow for hybridization. The probe consists of a fluorescent or radioactive marker. A sheet of photographic film is then placed over the membrane. After a period of incubation, the radioactivity darkens the film, revealing the location of the gene of interest. See the process flow below.

The development of Southern blotting led to similar techniques such as Northern blotting to detect the presence of mRNA (hence gene expression) and Western blotting to detect protein. PCR has mostly replaced blotting techniques due to its speed, accuracy and efficiency. 

Sequencing 

Historically, the most common method of DNA sequencing is the dideoxynucleoside termination sequencing or simply, Sanger sequencing method. The first step in the method is to amplify the DNA of interest using chain-termination PCR. This type of PCR works the same way are standard PCR, except with the addition of modified nucleotides called dideoxynucleotides (ddNTPs).  During PCR, the DNA is denatured and a primer added to the 5’end of the template. Four PCR reactions are set up with each containing DNA polymerase, the four nucleotides (dNTP) and four modified nucleotides (ddNTP), i.e. – ddATP, ddTTP, ddGTP, and ddCTP. The modification is characterized by the removal of the OH functional group from the 3-carbon position of the dNTP. This prevents DNA polymerase from adding additional nucleotides during DNA replication. Hence, termination occurs. Another modification is that each ddNTP is radiolabeled. 

The result of the PCR reaction in each vessel is the production of millions to billions of copies of DNA fragments of the sequence of interest, terminated at random lengths based on the ddNTP present in the vessel.

The fragments are then separated by gel electrophoresis and the sequence read from the bottom of the plate (smallest to largest fragment).

 Sanger sequencing has mostly been replaced by next generation sequencing which is capable of sequencing much larger genome sizes.

Reference: Klug, W. S., Cummings, M. R., Spencer, C. A., & Palladino, M. A. (2015). Concepts of genetics. New York, N.Y: Pearson Education Inc.