Key Technologies

List key technologies enabling modern uses of biology

Biotechnology is the use of biological agents for technological advancement. Biotechnology was used for breeding livestock and crops long before the scientific basis of these techniques was understood. Since the discovery of the structure of DNA in 1953, the field of biotechnology has grown rapidly through both academic research and private companies. The primary applications of this technology are in medicine (production of vaccines and antibiotics) and agriculture (genetic modification of crops, such as to increase nutrient content). Biotechnology also has many industrial applications, such as fermentation, the treatment of oil spills, and the production of biofuels (Figure 1).

The left side of this image is an old black and white photo of a mailbox plastered with an advertisement reading “Penicillin cures gonorrhea in four hours. See your doctor today.” The right side of the image shows a petri dish streaked with bacteria. Bacteria grow everywhere on the plate except where discs containing antibiotic have been placed. These areas are completely devoid of bacterial growth

Figure 1. Antibiotics are chemicals produced by fungi, bacteria, and other organisms that have antimicrobial properties. The first antibiotic discovered was penicillin. Antibiotics are now commercially produced and tested for their potential to inhibit bacterial growth. (credit “advertisement”: modification of work by NIH; credit “test plate”: modification of work by Don Stalons/CDC; scale-bar data from Matt Russell)

In this outcome, we will learn about some modern technologies used in biology today.

Learning Objectives

  • List basic techniques to manipulation genetic information (DNA and RNA)
  • Recognize technologies used for molecular, cellular, and reproductive cloning
  • Understand the basics of genetic engineering

Manipulating Genetic Material

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. The two strands can be separated by exposure to high temperatures (DNA denaturation) and can be reannealed by cooling. The DNA can be replicated by the DNA polymerase enzyme. Unlike DNA, which is located in the nucleus of eukaryotic cells, RNA molecules leave the nucleus. The most common type of RNA that is analyzed is the messenger RNA (mRNA) because it represents the protein-coding genes that are actively expressed. However, RNA molecules present some other challenges to analysis, as they are often less stable than DNA.

DNA and RNA Extraction

To study or manipulate nucleic acids, the DNA or RNA must first be isolated or extracted from the cells. Various techniques are used to extract different types of DNA (Figure 2). Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as degradation of unwanted molecules and separation from the DNA sample). Cells are broken using a lysis buffer (a solution which is mostly a detergent); lysis means “to split.” These enzymes break apart lipid molecules in the cell membranes and nuclear membranes. Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases (RNAses) that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. The DNA samples can be stored frozen at –80°C for several years.


Figure 2. This diagram shows the basic method used for extraction of DNA.


Photo shows an agarose gel illuminated under UV light. The gel is nine lanes across. Each lane was loaded with a sample containing DNA fragments of differing size that have separated as they travel through the gel, from top to bottom. The DNA appears as thin, white bands on a black background. Lanes one and nine contain many bands from a DNA standard. These bands are closely spaced toward the top, and spaced farther apart further down the gel. Lanes two through eight contain one or two bands each. Some of these bands are identical in size and run the same distance into the gel. Others run a slightly different distance, indicating a small difference in size.


Gel Electrophoresis


Amplification of Nucleic Acid Fragments by Polymerase Chain Reaction





Hybridization, Southern Blotting, and Northern Blotting

Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting (Figure 5). The nucleic acid fragments that are bound to the surface of the membrane can then be probed with specific radioactively or fluorescently labeled probe sequences. When DNA is transferred to a nylon membrane, the technique is called Southern blotting, and when RNA is transferred to a nylon membrane, it is called northern blotting. Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression. Note that “Southern” blotting is capitalized but no other type of blotting; Southern blotting is named after the scientist who pioneered this technique, Edwin Southern. The other types of blotting were named in reference to the original Southern blot.

In Southern blotting, DNA is separated on the basis of size by agarose gel electrophoresis. The fragments run through the gel from top to bottom. In the gel shown in this figure, there are so many DNA fragments they appear as a smear in each lane. The DNA from the gel is transferred to a nylon membrane. To do so, the gel is sandwiched between filter paper and the membrane and placed in hybridization buffer. Paper towels above the gel wick up the moisture and assist in the transfer. The nylon membrane is then incubated with a radioactively or fluorescently labeled probe that is complementary to the sequence of interest. Discrete bands appear where the sequence of interest is located.

Figure 5. Southern blotting is used to find a particular sequence in a sample of DNA. DNA fragments are separated on a gel, transferred to a nylon membrane, and incubated with a DNA probe complementary to the sequence of interest. Northern blotting is similar to Southern blotting, but RNA is run on the gel instead of DNA. In western blotting, proteins are run on a gel and detected using antibodies.

DNA Sequencing


Part A shows a template DNA strand and newly synthesized strands that were generated in the presence of dideoxynucleotides that terminate the chain at different points to generate fragments of different sizes. Each dideoxynucleotide is labeled a different color. Part B shows a sequence readout that was generated after the DNA fragments were separated on the basis of size. The color of the fragment indicates the identity of the nucleotide at the end of a given fragment. By reading the colors in order, the DNA sequence can be determined.





Sanger’s genome sequencing has led to a race to sequence human genomes at a rapid speed and low cost, often referred to as the $1000 in one day sequence. Learn more by watching the Sequencing at Speed animation here.

Neanderthal Genome: How Are We Related?




Molecular Cloning


Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA of microorganisms such as E. coli. In cloning, the plasmid molecules can be used to provide a “folder” in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA, or a transgene, to differentiate it from the DNA of the bacterium, which is called the host DNA.

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly available restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease (Figure 1).

Recombinant DNA Molecules

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.


Cellular Cloning


Practice Question

Figure illustrates the steps in molecular cloning into a plasmid called a cloning vector. The vector has a lacZ gene, which is necessary for metabolizing lactose, and a gene for ampicillin resistance. Within the lacZ gene are restriction sites, sequences of DNA cut by a particular restriction enzyme. The DNA to be cloned and the plasmid are both cut by the same restriction enzyme. The restriction enzyme staggers the cuts on the two strands of DNA, such that each strand has an overhanging single-stranded bit of DNA. On one strand, the sequence of the overhang is GATC, and on the other, the sequence is CTAG. These two sequences are complementary, and allow the fragment of foreign DNA to anneal with the plasmid. An enzyme called ligase joins the two pieces together. The ligated plasmid is then transformed into a bacterial strain that lacks the lacZ gene and is sensitive to the antibiotic ampicillin. The bacteria are plated on media containing ampicillin, so that only bacteria that have taking up the plasmid (which has an ampicillin resistance gene) will grow. The media also contains X-gal, a chemical that is metabolized in the same way as lactose. Plasmids lacking the insert are able to metabolize X-gal, releasing a dye from X-gal that turns the colony blue. Plasmids with the insert have a disrupted lacZ gene and produce white colonies. Thus, colonies containing the cloned DNA can be selected on the basis of color.

Figure 1. This diagram shows the steps involved in molecular cloning.


  1. There will be no colonies on the bacterial plate.
  2. There will be blue colonies only.
  3. There will be blue and white colonies.
  4. The will be white colonies only.

Reproductive Cloning

Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism. Most multicellular organisms undergo reproduction by sexual means, which involves genetic hybridization of two individuals (parents), making it impossible for generation of an identical copy or a clone of either parent. Recent advances in biotechnology have made it possible to artificially induce asexual reproduction of mammals in the laboratory.

Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization of the egg occurring; this is a form of asexual reproduction. An example of parthenogenesis occurs in species in which the female lays an egg and if the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male. The unfertilized egg is called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into adults.

Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results. The zygote nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis for reproductive cloning. Therefore, if the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor. Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.

The first cloned animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for seven years and died of respiratory complications (Figure 2). There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual. Since Dolly, several animals such as horses, bulls, and goats have been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities. There have been attempts at producing cloned human embryos as sources of embryonic stem cells, sometimes referred to as cloning for therapeutic purposes. Therapeutic cloning produces stem cells to attempt to remedy detrimental diseases or defects (unlike reproductive cloning, which aims to reproduce an organism). Still, therapeutic cloning efforts have met with resistance because of bioethical considerations.

Practice Question

To clone Dolly the sheep, a Scottish Blackface sheep was used as a cytoplasmic donor. Eggs from this sheep were extracted, and the nucleus removed. A Finn-Dorset sheep was used as the nuclear donor. Nuclei were extracted from mammary cells, and direct electric current was used to fuse the nuclear DNA with the donor egg. The egg was then allowed to divide to the blastocyst stage, in which a sphere of cells contains a cluster of cells on one side. The blastocyst was implanted in a surrogate mother, resulting in Dolly the sheep.

Figure 2. Dolly the sheep was the first mammal to be cloned. To create Dolly, the nucleus was removed from a donor egg cell. The nucleus from a second sheep was then introduced into the cell, which was allowed to divide to the blastocyst stage before being implanted in a surrogate mother. (credit: modification of work by “Squidonius”/Wikimedia Commons)


Genetic Engineering


Gene Targeting


Check Your Understanding



  1. Richard E. Green et al., “A Draft Sequence of the Neandertal Genome,” Science 328 (2010): 710–22.