Gene technologies allow the study and alteration of gene function

In a nutshell

Gene technologies let us cut out a gene from one organism and get another organism to read it, because the genetic code and the machinery of transcription and translation are universal.

This subtopic covers three things built on that idea: recombinant DNA technology (moving a gene into a host and getting it expressed), using labelled DNA probes to screen a person's DNA for specific alleles, and genetic fingerprinting using VNTRs.

Assumed knowledge: DNA and protein synthesis, Using genome projects.

Core content

What recombinant DNA technology is

Recombinant DNA technology transfers a fragment of DNA from one organism, or species, to another.

The transferred DNA can be read by the recipient because the genetic code is universal, and transcription and translation work the same way in all organisms. The recipient is called a transgenic organism, and its DNA (from two different sources) is recombinant DNA.

The workflow is: make a fragment of the desired gene, amplify it, insert it into a vector, transform host cells, and identify the ones that took it up.

Making a DNA fragment (three methods)

There are three ways to obtain a fragment containing the desired gene.

• Reverse transcriptase, an enzyme that builds complementary DNA (cDNA) from an mRNA template. You extract the mRNA for the gene from a cell that is actively making that protein, then copy it back into DNA.
• Restriction endonucleases, which cut a fragment containing the gene out of DNA by cutting at a specific recognition sequence.
• A gene machine, which synthesises the DNA fragment chemically from the known base sequence.

cDNA and gene-machine DNA have no introns. This matters because a prokaryote such as E. coli cannot splice out introns (it has no spliceosome), so a gene taken straight from eukaryotic DNA with its introns would not be expressed correctly in a bacterial host.

Amplifying the fragment: PCR (in vitro)

The polymerase chain reaction (PCR) amplifies a DNA fragment in vitro (outside a living cell), doubling the amount of DNA each cycle. One cycle has three stages:

1. Denaturation: the DNA is heated (around 95°C) so the hydrogen bonds between the two strands break and the DNA becomes single-stranded.
2. Annealing: the mixture is cooled (around 55°C) so primers bind (anneal) to complementary sequences at the ends of the target strands.
3. Synthesis: a DNA polymerase (a heat-stable one, so it survives the denaturation temperature) joins free DNA nucleotides onto each primer, building a new complementary strand.

Primers are short, single-stranded pieces of DNA with a base sequence complementary to the start of the target strand. They matter for two reasons:

• DNA polymerase cannot start a strand from scratch; it can only add nucleotides to an existing strand, so a primer gives it a starting point.
• Two different primers are needed, one complementary to the start of each of the two strands, because the two strands are copied in opposite directions.

Still don't get it? · why PCR needs two different primers

Imagine you are copying out both sides of a torn page, and your pen only ever writes left to right. You cannot start writing in the middle of nowhere: you need a marker showing where each copy begins. And because the two sides read in opposite directions, the marker for the top side sits at one end and the marker for the bottom side sits at the other end. One marker will not do for both.

Now the exam version. The DNA has two strands running in opposite directions, and DNA polymerase can only build a strand in one direction and only by extending from an existing piece. So each strand gets its own primer, a short single-stranded piece of DNA with a base sequence complementary to the start of that strand. That is why PCR uses two different primers, not one.

Amplifying the fragment: transformed host cells (in vivo)

The alternative to PCR is to put the gene into host cells and let the cells copy it as they divide. Several steps prepare and deliver the gene.

Add promoter and terminator regions. Before insertion, a promoter and a terminator region are attached to the fragment.

• The promoter is where RNA polymerase binds to start transcription, so the gene is actually expressed in the host.
• The terminator signals RNA polymerase to stop transcription at the end of the gene.

Insert the fragment into a vector. A vector (commonly a plasmid) carries the gene into the host cell.

• The same restriction endonuclease is used to cut both the fragment and the plasmid, so both are left with complementary sticky ends (short exposed lengths of unpaired bases).
• The sticky ends of the fragment base-pair with those of the plasmid, and DNA ligase joins them by reforming the phosphodiester bonds in the sugar-phosphate backbone, making a recombinant plasmid.

Still don't get it? · why both must be cut with the same restriction enzyme

Think of tearing a sheet of paper in a jagged line, then trying to fit two torn pieces back together. Only pieces torn along the same jagged line slot into each other, because each bump on one side matches a notch on the other. Pieces torn along different lines will not line up.

A restriction endonuclease cuts DNA only at its own specific recognition sequence, and it makes a staggered cut that leaves a short single-stranded overhang: a sticky end. If you cut the gene and the plasmid with the same enzyme, both sticky ends have the same, complementary base sequence, so they base-pair together.

The exam point students miss: the sticky ends fit because they have complementary base sequences, not because they are the same "shape". Say complementary bases, then let DNA ligase join the backbones.

Transformation. The recombinant plasmid is introduced into a host cell, for example a bacterium. This uptake of the vector is called transformation (it is helped by, for example, calcium ions and a heat shock, which make the membrane more permeable).

Marker genes: finding the transformed cells

Not every host cell takes up the vector, and not every vector is recombinant, so you need a way to pick out the cells that took up the gene.

A marker gene is inserted into the vector alongside the desired gene. It lets you identify which cells are transformed, for example by giving them antibiotic resistance, or making them fluoresce under UV light.

(You are not required to recall specific marker genes for a written paper; you do need to explain what a marker gene is for.)

Evaluating recombinant DNA technology

The spec expects you to evaluate the ethical, financial and social issues, and to balance the humanitarian benefits against the opposition, in agriculture, industry and medicine.

A good evaluate answer uses both sides and reaches a supported judgement relevant to the specific use in the question.

Relating recombinant DNA technology to gene therapy

Gene therapy uses recombinant DNA technology to supplement or replace a faulty allele with a functioning one.

• Somatic gene therapy targets body cells, so the change is not passed on to offspring. Because treated cells are replaced over time, the effect is temporary and treatment must be repeated.
• Germ-line gene therapy targets gametes or early embryos, so the change would be inherited. It is currently not permitted in humans.

Locating specific alleles: DNA probes and hybridisation

A DNA probe is a short, single-stranded piece of DNA with a base sequence complementary to a specific target allele, and it is labelled so it can be detected.

• A radioactively labelled probe is detected by exposing X-ray film (autoradiography).
• A fluorescently labelled probe is detected under UV light.

To use a probe, the target DNA is first made single-stranded (heat or alkali breaks the hydrogen bonds). The probe then hybridises: it binds by complementary base pairing to the target sequence only if that sequence is present. Wherever the label then shows up, the allele is present.

A DNA microarray carries many different probes on fixed spots, so thousands of alleles can be screened at once.

Screening, genetic counselling and personalised medicine

Labelled probes let clinicians screen a patient's DNA for alleles linked to heritable conditions, to drug responses, or to future health risks.

• Genetic counselling uses screening results to advise individuals and families on the risk of having or passing on a genetic condition, so they can make informed decisions.
• Personalised medicine uses a person's genetic information to choose the drug and dose most likely to work for them, since people with different alleles respond differently to the same drug.

You should be able to evaluate information about screening for genetically determined conditions and drug responses (for example, weighing early diagnosis against anxiety, insurance implications and false results).

Genetic fingerprinting and VNTRs

A genome contains many variable number tandem repeats (VNTRs): short base sequences in non-coding DNA that are repeated, with the number of repeats varying between individuals. The probability of two individuals having the same set of VNTRs is very low (identical twins aside), which is what makes fingerprinting reliable.

Genetic fingerprinting analyses DNA fragments that have first been cloned (copied) by PCR. The method:

1. Cut the DNA with restriction endonucleases at recognition sites either side of the VNTRs, giving fragments whose length depends on the number of repeats.
2. Separate the fragments by gel electrophoresis.
3. Transfer the fragments to a nylon membrane and make them single-stranded.
4. Apply a labelled DNA probe complementary to the VNTR sequences, which hybridises to the fragments.
5. Detect the bound probe (X-ray film for radioactive, UV for fluorescent), giving a pattern of bands: the genetic fingerprint.

Reading a gel: how electrophoresis separates fragments

Gel electrophoresis separates DNA fragments by length (mass).

• DNA is negatively charged (because of the phosphate groups), so when an electric current is applied the fragments move towards the positive electrode (anode).
• The gel acts as a sieve: smaller (shorter) fragments move faster and travel further; larger fragments are held back and stay near the wells.

Because fragment length depends on the number of VNTR repeats, individuals with different repeat numbers give bands at different positions.

Uses of genetic fingerprinting include forensic science (matching crime-scene DNA to a suspect), medical diagnosis, and animal and plant breeding, as well as determining genetic relationships (such as paternity) and the genetic variability within a population.

Worked examples

Worked calculation: amplifying DNA by PCR

PCR doubles the number of DNA molecules each cycle, so from one starting molecule the number of copies after $n$ cycles is:

$N = 2^{n}$

How many molecules are produced from a single DNA molecule after 25 cycles?

$N = 2^{25} = 33\,554\,432 \approx 3.4 \times 10^{7}$

So one molecule becomes roughly 34 million copies. If you start with $x$ molecules rather than one, use $N = x \times 2^{n}$.

The chart below shows the first eight cycles, making the exponential doubling visible.

Model long-answer: describe how a recombinant plasmid is made and used to produce a human protein in bacteria

A full-mark answer moves through these linked points in order:

1. Isolate the gene, for example by using reverse transcriptase to make cDNA from the mRNA (no introns), or by cutting it out with a restriction endonuclease.
2. Attach a promoter and a terminator region so the gene can be transcribed in the host.
3. Cut the gene and the plasmid with the same restriction endonuclease so both have complementary sticky ends.
4. Join the gene into the plasmid using DNA ligase, which reforms the phosphodiester bonds, giving a recombinant plasmid.
5. Transform the bacteria by taking up the recombinant plasmid.
6. Use a marker gene to identify the cells that have taken up the gene.
7. Culture the transformed cells so they divide and express the gene, producing the protein.

Common exam mistakes

• Saying sticky ends join because they have the same shape. They join because they have complementary base sequences that base-pair; say "complementary bases".
• Forgetting that the gene and plasmid must be cut with the same restriction enzyme, which is why their sticky ends are complementary.
• Saying DNA ligase cuts DNA, or confusing it with a restriction enzyme. Ligase joins fragments by reforming phosphodiester bonds.
• Naming the wrong enzyme for making cDNA. It is reverse transcriptase; "restriction endonuclease" is a common wrong answer.
• In PCR, saying the strands are separated by an enzyme (helicase, or a restriction enzyme). They are separated by heat breaking the hydrogen bonds.
• Being unable to say why two primers are needed, or calling a primer just "specific" without saying its base sequence is complementary to the start of a strand.
• Claiming PCR is "conservative", or that PCR separates DNA fragments. PCR amplifies (copies) DNA; electrophoresis is what separates fragments.
• Describing a DNA probe without saying it is single-stranded and complementary to the target allele, and muddling up the words allele, base and probe.
• In gel electrophoresis, forgetting that DNA moves because it is negatively charged, and that fragments separate by length/mass with smaller fragments travelling further. Do not confuse it with chromatography.
• Saying VNTRs are in coding DNA. They are in non-coding DNA, and it is the number of repeats that varies between individuals.
• In a long fingerprinting answer, starting with lots of PCR detail and getting the order of steps wrong (for example separating strands before electrophoresis, or adding the probe too early).

Key definitions

• Recombinant DNA: DNA made by combining (joining) DNA fragments from two different organisms or species.
• Transgenic organism: an organism that contains recombinant DNA transferred from a different species.
• Restriction endonuclease (restriction enzyme): an enzyme that cuts DNA at a specific recognition sequence.
• Sticky ends: the short, exposed sequences of unpaired bases left when a restriction enzyme makes a staggered cut in DNA.
• DNA ligase: an enzyme that joins DNA fragments together by reforming phosphodiester bonds.
• Vector: a carrier, such as a plasmid or a virus, used to transfer a DNA fragment into a host cell.
• Reverse transcriptase: an enzyme that produces complementary DNA (cDNA) from an mRNA template.
• Polymerase chain reaction (PCR): a method of amplifying DNA in vitro to produce many copies of a DNA fragment.
• Primer: a short, single-stranded piece of DNA with a base sequence complementary to the start of a target DNA strand.
• Transformation: the uptake of a vector carrying recombinant DNA by a host cell.
• Marker gene: a gene inserted into a vector alongside the desired gene, used to identify cells that have taken up the recombinant DNA.
• DNA probe: a short, single-stranded piece of labelled DNA with a base sequence complementary to a specific target allele.
• DNA hybridisation: the binding of a single-stranded DNA probe to a complementary single-stranded target sequence by base pairing.
• Variable number tandem repeats (VNTRs): short base sequences in non-coding DNA that are repeated a number of times, with the number of repeats varying between individuals.
• Genetic fingerprinting: a technique that produces a pattern of DNA fragments (from VNTRs) that is unique to an individual (except identical twins).

Specification

• I can explain that recombinant DNA technology transfers a fragment of DNA between organisms or species, and that it works because the genetic code and the mechanisms of transcription and translation are universal.
• I can describe how DNA fragments are produced using reverse transcriptase (cDNA from mRNA), restriction endonucleases, and a gene machine.
• I can describe the principles of PCR as an in vitro method, and the culture of transformed host cells as an in vivo method, of amplifying DNA fragments.
• I can explain the addition of promoter and terminator regions to a DNA fragment.
• I can describe the use of restriction endonucleases and ligase to insert a fragment into a vector, and the transformation of host cells using these vectors.
• I can explain the use of marker genes to detect genetically modified cells or organisms.
• I can evaluate the ethical, financial and social issues of recombinant DNA technology in agriculture, industry and medicine, and relate the technology to gene therapy.
• I can explain the use of labelled DNA probes and DNA hybridisation to locate specific alleles and to screen patients for heritable conditions, drug responses or health risks, and the use of this in genetic counselling and personalised medicine.
• I can explain the biological principles of genetic fingerprinting using VNTRs, interpret gel electrophoresis results, and explain its uses in forensic science, medical diagnosis and animal and plant breeding.

Related notes

• DNA and protein synthesis
• The alteration of the sequence of bases in DNA
• Gene expression is controlled
• Using genome projects