Lesson Explainer: Molecular Technologies Biology

In this explainer, we will learn how to outline the processes of gene machines and DNA hybridization and recall some applications of these molecular technologies.

DNA is a huge molecule! In humans, the complete set of DNA is about 3.2 billion nucleotide pairs, and in chimpanzees, it is about 3.1 billion nucleotide pairs. It might surprise you that humans share about of their DNA with chimpanzees! How we came to this number has everything to do with molecular technology.

Molecular technology is a broad term that involves different laboratory techniques to study or modify DNA, RNA, or proteins. There are different types of molecular technologies available to help us advance medicine, agriculture, forensics, and many other fields. For example, you may recall that recombinant DNA involves combining two sources of DNA to create new genetic information. This is a type of molecular technology that can be used to manufacture insulin to treat diabetes when we place the gene for insulin into bacteria.

In this explainer, we will go through a few examples of molecular technology, namely bioinformatics, gene machines, and DNA hybridization.

Humans share of their DNA with chimpanzees. How do we know this?

One way to see if two organisms are evolutionarily related is to study their morphology, or physical characteristics, and look for similarities. The bone structure of our hand is more similar to that of a chimpanzee and less similar to that of a frog. So, on this basis, we can say that we are more closely related to chimpanzees than to frogs. Beside morphology, another way to examine evolutionary relationships between organisms is to study their molecular similarities, which can be done by comparing DNA or protein sequences.

Bioinformatics is a field of study that combines biology and computer science, making it possible to analyze huge amounts of biological data. The genome of humans and that of chimpanzees are extremely massive, and comparing them manually would be nearly impossible! Using computers, we can line up these two sequences and see what is similar and what is different. An example of this is shown in Figure 1 below.

In the case of humans and chimpanzees, there is only about a difference in DNA sequences. This shows a strong evolutionary relationship between us and chimpanzees.

Bioinformatics can also be used to compare protein sequences between organisms. Insulin is a hormone that is involved in regulating sugar levels in the blood and is found in many organisms. You will recall that a protein is assembled as a sequence of amino acids, each of which can be represented by letters.

Over the course of evolution, the gene for insulin randomly accumulates mutations that can change this sequence of amino acids. Since this can take a lot of time, organisms that are more distantly related will have more differences in their protein sequences. You can see this in Figure 2 where there are more differences between chicken and human insulin than there are between the sequences for chimpanzee and human insulin. This suggests that humans and chimpanzees are more closely related than humans and chickens.

Bioinformatics has made it possible to uncover more about our evolutionary past and is a powerful example of molecular technology. However, suppose we are interested in making copies of the insulin gene in order to study it in the lab. How might we do this?

You will recall that when a protein is made in a cell, the nucleotide sequence in the DNA is transcribed into mRNA and that mRNA sequence is translated by a ribosome into a polypeptide chain, or protein sequence. When we want to synthesize a gene from a protein sequence, we can use technology to carry out this process but backward.

With a gene machine, it is possible to synthesize a gene by simply inputting the protein sequence into a computer! There are several steps to this process.

The first step is to select a protein of interest. In this example, we will choose the insulin protein from a chicken, a human, and a chimpanzee, as shown in Figure 3. For simplicity, let’s just focus on a smaller region of 14 amino acids in the sequence that shows the most differences. These sequences are highlighted in yellow in Figure 3 below.

The next step is to work out the DNA sequence from the protein sequence. You will recall that groups of three nucleotides, called codons, are translated from mRNA into individual amino acids using the genetic code. So, in order to determine the DNA sequence from the protein sequence, we must first determine the mRNA sequence. You can see the result in Figure 4 below.

Now that we have our DNA sequence for these insulin segments, in the next step, we can input this sequence into the gene machine. The gene machine then synthesizes short molecules of DNA called oligonucleotides that can be used to assemble the full-length gene. Oligonucleotides are short pieces of synthetically produced DNA or RNA that are typically single stranded. They can have many applications in molecular technology, and in gene machines, they can be joined together to form the full-length gene.

The full-length gene can be assembled by a gene machine because the oligonucleotides are overlapping. One oligonucleotide will be assembled in the – direction just as the gene sequence is entered, while another oligonucleotide will be assembled in the opposite direction. The oligonucleotides can then act as a template for DNA synthesis using a specialized technique called polymerase chain reaction (PCR). During this step, double-stranded DNA is made from the single-stranded oligonucleotides. You can see all of this in Figure 5 below.

The basis of the technology of gene machines comes from Har Gobind Khorana, who was the first person to assemble a synthetic gene in the 1970s. The overall steps for producing the DNA sequence of a gene from a protein sequence using a gene machine are indicated in Figure 6 below.

Gene machines can be used to make any gene you want! Importantly, they are lacking the introns, or noncoding DNA, that may be found in the naturally occurring gene. They can also be useful in studying how proteins work. For example, we could change a single nucleotide that would change an amino acid in the insulin gene and examine the effect this has on the protein’s function.

Now, let’s look at an important property of DNA called hybridization that can be used in molecular technology.

DNA is a double-stranded molecule that is joined through the hydrogen bonding between complementary nucleotides on opposing strands, as shown in Figure 7 below. When DNA is heated to high temperatures (usually –), the hydrogen bonds that join the two strands together can weaken and separate, forming two single strands. Single-stranded DNA is unstable. When cooled rapidly (–), it can stick, or anneal, to its complementary strand by hydrogen bonding, once again forming double-stranded DNA.

The sources of the two strands of DNA do not need to be the same, so one can be from human DNA and the other from chimpanzee DNA. RNA can also anneal to a single strand of complementary DNA. The ability to mix single-stranded DNA from two sources or to mix single-stranded DNA and RNA can be used to produce a hybrid molecule. This is called hybridization.

During hybridization, sometimes the two sequences are not totally complementary. In this case, those noncomplementary nucleotides will not form hydrogen bonds or anneal to each other. A high number of complementary nucleotides, and therefore hydrogen bonds, makes for a stronger interaction between the two sequences that requires a higher temperature to split apart. This property can be used to evaluate the similarity in sequence between two molecules of DNA.

Going back to our example of the chicken, human, and chimpanzee insulin genes, we can apply this idea of hybridization to see how similar the sequences are to one another. By heating the DNA for a segment of insulin from both chicken and human DNA and then rapidly cooling the mixture, two single strands of DNA will hybridize to each other. You can see this in Figure 8 below.

The above example showed how human and chicken insulin DNA hybridize, and the same can be done for human and chimpanzee DNA. The hybridizations for both human and chicken and human and chimpanzee are shown in Figure 9 below. We can see that there is more hydrogen bonding between the human and chimpanzee insulin DNA compared to the chicken and human insulin DNA. Therefore, a higher temperature would be needed to separate the human and chimpanzee DNA. This higher degree of hybridization also indicates that human insulin is more closely related to chimpanzee insulin compared to chicken insulin.

Example 4: Using DNA Hybridization to Measure Evolutionary Relationships

DNA hybridization can be used to help determine the evolutionary relationship between two species, the process of which has been simplified and outlined in the diagram provided. When DNA hybridizes, it will form hydrogen bonds between any base pairs that are complementary. An X on the diagram indicates a hydrogen bond has not formed.

Which of the following is an assumption scientists make when using this technique?

1. DNA taken from two different species will form more hydrogen bonds if those two species are closely related.
2. DNA taken from two different species will form fewer hydrogen bonds if those two species are closely related.
3. The number of hydrogen bonds formed between hybridized DNA does not indicate how closely related those species are.

Answer

DNA is a double-stranded molecule composed of nucleotides that associate with each other based on complementary base-pairing rules. This association is through hydrogen bonding and is what joins the two DNA strands together.

This hydrogen bonding can be broken by heating the DNA molecule to high temperatures. By breaking these hydrogen bonds, the two single strands of DNA separate. Upon cooling, these two strands can once again hydrogen bond through their complementary bases to form the double-stranded molecule. This process is called annealing. We can join two different sources of DNA (or RNA) together to form a hybrid.

If two DNA sequences are similar to each other, then they will form hydrogen bonds between their complementary nucleotides between the strands. The more similar the sequences are, the more hydrogen bonds will form between the two strands.

Over the course of evolution, a species can take on multiple random mutations in their DNA. Since this can take a lot of time, scientists make the assumption that organisms that are more distantly related will have more differences in their DNA. So, if we compare the DNA sequence of a human and a chicken (distantly related) to that of a human and a chimpanzee (closely related), we will see more hydrogen bonding in the human-and-chimpanzee DNA hybrid:

Therefore, during DNA hybridization, DNA taken from two different species will form more hydrogen bonds if those two species are closely related.

Hybridization can be used in different applications of molecular technology. An example is microarrays. These are tiny microchips with multiple spots that each contain a unique oligonucleotide specific to one gene. A single microarray chip can have thousands of spots and, therefore, can be used to assess thousands of different genes. Microarrays can be used to study gene expression to see if different tissues express more mRNA for a huge number of genes.

The mRNA from different tissues can be labeled with different fluorescent markers. For example, the mRNA from normal tissue can be labeled green, and the mRNA from a tumor can be labeled red. When these samples are combined and loaded into a microarray to hybridize, each spot will fluoresce depending on the number of green or red mRNAs present. So, if the tumor has more of a particular mRNA compared to the healthy tissue, then the spot for that corresponding gene will be more red. This is shown in Figure 10.

Let’s recap some of the key points we have covered in this explainer.