2.4- Gene Technology

What is PCR?

Polymerase Chain Reaction

It’s used to amplify target DNA sequences that are present in a DNA source.

It can generate billions of copies of a DNA sample within hours.

What does PCR require?

• A DNA sample that includes the selected region for application

• Synthesis of primers that are complementary to the sequence at the start of each strand of the region to be amplified

• Taq DNA polymerase extracted from thermophlic bacteria

• Free deoxyribonucleotides

What are primers?

A short single stranded chain of around 20 DNA nucleotides with a complementary base sequence to one end of the fragment being copied

Outline the process of PCR

It involves the repetition of a 3 step cycle in a thermocycler:

• Strand Separation- DNA to be amplified is heated 95°C to separate the DNA strands by breaking hydrogen bonds

• Primer Binding- Cool the solution to 50°C to allow annealing of DNA primer to the start of each strand of the complementary DNA region. It’s cooled to allow H bonds to form between complementary bases

• Strand Synthesis- The solution is heated to 70°C which is the optimum temp for taq DNA polymerase. The heat-stable DNA polymerase builds a new strand alongside each template strand, forming 2 identical double stranded DNA molecules

This can be repeated & each cycle lasts for around 2 mins & doubles the number of DNA molecules

insert diagram from study guide pg 30

What are the uses of PCR?

Used by forensic scientists to study minute samples of DNA- using root hairs etc

Used by archaeologists to study minute samples of DNA- investigating extracted DNA from bones of extinct species to allow them to find out how closely related they are to living species

Used in genetic fingerprinting if a sample needs to be amplified

What are restriction enzymes?

They cut the sugar phosphate backbone of DNA at specific nucleotide base sequences.

• They may make straight cuts which leave blunt edges

• Or they may make staggered cuts which leave sticky ends (unpaired bases to which another DNA fragment with with the complementary code could bind)

What is a DNA probe?

A short single strand of DNA with a complementary base sequence to the target DNA trying to be located

How can probes be labelled?

Radioactive labelling: detected using X-ray film

Fluorescent labelling: detected using UV light or a laser scanner

The probes are used to identify specific gene sequences

What are genetic markers?

DNA sequences with known locations on chromosomes, which are points of variation & so used to identify individuals

How can differences in nucleotide sequences be identified?

Assessing the differences in nucleotide sequences can be a measure of genetic variation

Genetic markers to do this include:

• Microsatellite repeat sequences (MRSs)-

Different people have different numbers of repeats in their MRSs (eg CACACACACA)

Therefore, different people have different sized MRSs.

MRSs are different in all individuals except identical twins

Done during genetic fingerprinting

• Single nucleotide polymorphisms (SNPs)-

These are sites in the genome where a single nucleotide has be substituted

SNPs represent different allelic differences & some are linked with genetic disease

What is mircroarray technology?

Used to detect gene expression/ gene sequence variation of thousands of genes in an individual by using microarrays which consist of many different DNA probes immobilised on a solid surface.

Each spot of the array contains many molecules of a particular probe & different spots possess probes complementary to different known genes.

Outline the process of using microarrays in genotype testing

• DNA is hybridised to a microarray chip

• Sample DNA bound to a probe on the chip which can produce a signal that can be fluorescent or chemiluminescent

• A computer analyses the chip to calculate the strength of the signal produced at each spot, with the strength of the signal representing the level of gene expression in the individual

The principle of this is the hybridisation between the DNA probe & any complementary single stranded DNA from the sample.

If a DNA fragment hybridises with a probe, a colour change occurs.

Outline the process of using microarrays in testing for gene expression

The mRNA is extracted from the tissue & used to make single stranded cDNA (catalysed by reverse transcriptase).

The DNA molecules used are labelled with a fluorescent tag

The labelled cDNA produced is added to the microarray & hybridises to any complementary probes

Hybridisation will indicate that a gene is expressed

What is genetic testing used for?

Identifying:

• Genotypes:

Defective alleles (often SNPs) have distinctive base sequence

Important to identify for diagnosing genetic disease or a carrier

• Gene expression:

If a gene is expressed, then mRNA is transcribed & a protein is synthesised. The presence of mRNA in the cytoplasm indicates a gene is being expressed

Important in the diagnosis of cancer where genes become activated & lead to uncontrolled cell division

How is genetic fingerprinting done?

• Restriction endonucleases cut chromosomal DNA

• Fragments separate according to size (using gel electrophoresis) to produce unique profiles

• DNA probes locate specific DNA fragments

Outline the steps for genetic fingerprinting

Extracting DNA

If sample is small, PCR can be used.

In diploid eukaryotes, it will contain the DNA of 2 sets of chromosomes (1 maternal & 1 paternal)

Digestion

Restriction enzymes cut out repeated sequences

The enzymes are chosen so they cut at specific base sequences at either side of the MRSs.

Repeat sequences remain intact. If there’s a large number of repeats, then the fragments will be long & vice versa.

Separation:

The mixture of fragments with varying lengths is separated by gel electrophoresis.

Smaller fragments will move further

Transfer fragments onto a nylon sheet:

Fragments are heat treated to make them single stranded & then transferred onto a nylon sheet by pressing it against the gel (Southern blotting)

Hybridisation:

The separated DNA fragments aren’t visible so radioactive probes are hybridised onto the fragments containing the complementary repeat sequence

Detecting different repeat sequences:

The fragments with radioactive/fluorescent probes attached show up when the nylon membrane is overlaid with X ray/UV

insert diagram of gel electrophoresis

Explain the process of gel electrophoresis

• DNA is extracted & cut into fragments by a restriction enzyme

• Mixture of DNA fragments are loaded into wells in a porous gel

• The gel floats in a buffer solution within a chamber between 2 electrodes

• Due to the phosphate ions, DNA has a negative charge. When an electric current is passed through the chamber, neg charged DNA fragments move towards the positive end

• Smaller fragments move fast & further

• The gel is treated with alkali to split the double stranded DNA.

• Single strands are then transferred to a nylon membrane by southern blotting.

• Exposing the nylon membrane to X-ray film & only the DNA bands which hybridised with the radioactive probe will affect the X-ray film

Summarise the main steps in gene transfer

1. Isolation- to obtain DNA fragment with required gene by reverse transcription or by cutting out the gene

2. Insertion- insert gene into a vector (viruses & plasmids)

3. Transformation- vector introduces new gene into a host bacterial cell

4. Identification- identify bacteria which have successfully taken up the gene

5. Cloning- culture genetically modified bacteria to produce the gene on a large scale

What are the methods of isolating a gene?

1. Use restriction endonuclease to cut the gene out of chromosomal DNA

DNA is cut into fragments with appropriate enzyme which cuts either side of the gene

Fragment with required gene identified with complementary probe

A restriction enzyme that produces sticky ends is more useful & if the same one is used to open a vector, the exposed bases of both are complementary & will more readily attach through base pairing

Will need to have its introns removed

2. Use reverse transcriptase to produce DNA from mRNA

mRNA is obtained from cells where the gene concerned is actively synthesising protein

Reverse transcriptase uses the mRNA as a template to produce cDNA from free DNA nucleotides

Double stranded DNA is made from the cDNA using DNA polymerase

Genes synthesised from mRNA won’t have introns so it will be able to direct protein synthesis when inserted into bacteria

insert figure 28 & 29 from pg 40 of study guide

What are the methods of inserting a gene into a vector?

Bacterial plasmids:

Plasmid is cut open using the same restriction enzyme used to cut the DNA fragment out of the donor DNA

The sticky ends of each type of DNA contain complementary base sequences

Plasmid DNA & gene DNA anneal. H bonds readily form between the complementary bases of the sticky ends

DNA ligase catalyses the formation of phosphodiester bonds between the sugar phosphate backbones of the plasmid & the gene DNA

The gene has now been spliced into the plasmid

DNA with foreign DNA inserted is called recombinant DNA, now the plasmid is a recombinant plasmid

Viruses:

Already adapted to insert their genetic material into a host cell by injecting their DNA into a bacterium

insert figure 30 on pg 41 of study guide

What is a vector?

A delivery tool to carry the gene into the host cell where it can be replicated & expressed

How is a vector inserted into a host cell?

Bacterial cells naturally take up plasmids but they do this more readily when induced.

Cells are incubated with calcium ions & treated with a heat shock (rapid temp from 0-40)

The bacteria will temporarily become more permeable (as material making up cell wall is separated) to allow plasmids to enter the cells

The modified bacteria is transgenic

Not all will contain the gene so they need to be identified

How is the transgenic bacteria identified?

Through the use of a marker gene (such as antibiotic resistance or a fluorescent marker gene)

For antibiotic resistant markers:

Insert gene to make the bacteria resistant to tetracycline & ampicillin

Appropriate restriction enzyme cut in the middle of the tetracycline gene

Insert isolated DNA fragment to be cloned

The resistance to tetracycline is disrupted by DNA fragment so the gene won’t create the functional protein

Grow bacteria on agar

• Those that failed to take up plasmids are sensitive to ampicillin & tetracycline

• Those that take up the original plasmids are resistant to both ampicillin & tetracycline

• Those that take up the recombinant plasmids are resistant to ampicillin but not tetracycline

insert figure 31 on pg 42 of studt guide

Using fluorescent marker genes:

Cut open plasmids using a restriction enzyme

Insert genes for fluorescence & insulin using DNA ligase

Allow bacteria to take up plasmids

Culture bacteria on agar plate

Transgenic bacteria will glow under UV light

How is the transgenic bacteria cloned?

Bacteria are transferred to a sterile nutrient medium to increase in number (gene cloning)

As they grow, at intervals the culture solution is drawn off & the human proteins are purified

What are genetically modified microorganisms?

Bacteria, yeasts, or fungi whose genetic material has been altered, often using recombinant DNA technology or CRISPR, to introduce new, desirable traits

What substances can GM bacteria produce?

Bacteria have been genetically modified to produce human insulin, so no more allergic reactions occur as cow or pig insulin is no longer used

Also have been genetically modified to produce human growth hormone, cortisone or sex hormones

They can also be modified to produce chemicals such as enzymes, lung surfactant protein or interferon

What can GM viruses produce?

The herpes virus can have a gene inserted which codes for a protein that stimulates the immune system- can be used to attack skin cancer cells & make them more vulnerable to immune response

Bacteriophages can be modified to impair the bacterium’s ability to repair damaged DNA- making the bacteria more vulnerable to DNA damaging drugs

What are genetically modified organisms?

An organisms whose genetic material (DNA) has been altered using engineering techniques in a way that does not occur naturally by mating or reproduction

How is donor DNA inserted into animal cells?

Electroporation- cell membrane temporarily disrupted by a high voltage shock

Liposomes as vectors- can adhere to cell surface membrane & pass the DNA in a way like endocytosis

Viruses as vectors- adenovirus can be altered to not destroy host cells. They have RNA so when delivered, it’s copied to DNA & it’s incorporated into the host’s chromosome (useful in gene therapy)

Or by microinjection (DNA directly injected into nucleus of fertilised egg)

What is the purpose of transgenic animals?

encourage faster growth rate and better food quality traits

− produce substances of medical and pharmaceutical value

− use as models in human disease research