Gene Technology

Define the term recombinant DNA

DNA that is made by joining pieces from two or more different organisms

State what is meant by Genetic engineering/transfer

The manipulation of genetic material and is usually done to modify a characteristic in the host organism

Outline the stages of Gene transfer

1. The target gene is identified and may be cut from chromosomes, made from mRNA by reverse transcription or synthesised from nucleotides

2. Multiple copies of the gene are made using PCR

3. The gene is inserted into a vector which delivers the gene to the cells of the organisms

4. The vector takes the gene into the cell

5. The cells that have the new genes are identified and cloned

What does a genetic engineer need in order to perform gene transfer?

Enzymes - restriction endonucleases, ligase, reverse transcriptase

Vectors - viruses, plasmids

Genes coding for easily identifiable substances that can be used as markers

What is the function of the Polymerase Chain Reaction?

PCR is an in vitro method of cloning and amplifying a fragment of DNA

Outline the substances required for PCR

DNA fragments that’s going to be amplified

Primers (short sections of DNA)

DNA Taq polymerase

Free floating nucleotides

Describe the process of PCR or in vitro

1. Set up the reaction mixture

2. Heat the DNA fragments to 95 degrees in order to denature the DNA and break the hydrogen bonds

3. Cool the mixture at 65 degrees so that the primers can bind with the complementary bases so that a new strand of DNA can be formed (annealing)

4. Reheat the mixture to 72 degrees so that Taq polymerase can activate and make new complementary strands

5. Repeat. The number of DNA fragments is doubled each PCR cycle

Why is Taq Polymerase used during PCR?

It is thermophilic so it can withstand high temperatures and doesn’t denature

Why are primers needed for PCR?

They provide a starting point for Taq polymerase and they also prevent the two DNA strands from joining after they’ve been separated

What are recognition sites?

Sections of DNA where the base sequence has antiparallel base pairs

Outline the in vivo process of amplifying DNA fragments

1. The vector DNA is cut open by restriction endonucleases and now the vector DNA has sticky ends on each side

2. The DNA fragments with sticky ends that are complementary to the vector are bound to the Vector DNA by DNA ligase

3. The vector transfers the recombinant DNA to the host cells

4. Marker genes are inserted so that the transformed cells can be identified if they have recombinant DNA

What is gel electrophoresis?

A technique that is used to separate different molecules and is used a lot in the analysis of proteins and DNA

What is the function of gel electrophoresis?

To separate DNA fragments, nucleic acids and proteins according to their size and charge

State factors that affect the movement of charged molecules within the gel during gel electrophoresis

• Net overall charge (negative molecules move to the positive anode)

• Size (smaller molecules move faster than larger)

• Composition of the gel

Outline the process of Gel electrophoresis

1. DNA is amplified using PCR and labelled using fluorescent labels

2. Restriction enzymes cut DNA into fragments and then inserted into a well in the gel

3. The DNA moves in the gel towards the anode

4. DNA strands are then separated by heat or alkali

5. Probes are added to bind to specific DNA sequences and they can show up on X-ray film since they are radioactive

6. The alleles are then identified

How can different alleles be differentiated during gel electrophoresis?

Different alleles code for slightly different amino acid sequences

The charge of proteins will differ depending on the R group of amino acids present

The proteins can be separated according to the charge

What is a DNA probe?

A short single strand of nucleotides with a label attached. It is complementary to the base sequence of a particular gene in a DNA

The label may be fluorescent or radioactive

What is a plasmid?

A circular DNA structure found in bacteria. It carries DNA into the host cell

Why are plasmids used in genetic engineering?

They are easy to isolate and insert into DNA

They often have a gene for antibiotic resistance which can be used to identify bacteria that have taken up the plasmid

It’s double stranded so it can carry out prokaryotic and eukaryotic DNA

Why do promoters need to be transferred along with the desired gene?

Promoters control the expression of the gene. Transcription factors and RNA polymerase bind to the promoter sequence so transcription can occur

Describe how marker genes are used in gene technology

Marker genes produce a gene product or effect which is easily identifiable so they can confirm that the host cell has taken up the plasmid

What is the role of restriction endonucleases?

They cut viral DNA at specific site

They are part of a bacterium defence against pathogens, but can also be used for gene technology

How is reverse transcriptase used?

It is found in some viruses. It converts mRNA into single-stranded DNA which can produce double-stranded cDNA

Describe how DNA ligase is used in genetic engineering

DNA ligase joins the desired DNA fragment to the opened-up plasmid since they have complementary sticky ends and forms phosphodiester bonds between nucleotides to join the DNA

Outline how microarrays are used to analyse genomes

1. The DNA from each sample is denatured and then cut into fragments using restriction endonucleases

2. Each fragments is labelled with a fluorescent tag

3. The DNA is put onto a microarray that has thousand of probes which hybridise with their complementary genes

4. UV light is used to see where the DNA has hybridised to the probe and the genomes are identified and compared

How are microarrays used to asses gene expression?

mRNA is extracted from the cell and is converted back into cDNA using reverse transcriptase and DNA polymerase.

The DNA is tagged and hybridised with probes and the fluorescence at each spot shows how much mRNA is there aka the level of expression of gene

What does ‘Bioinformatics’ mean?

The collection, storage and analysis of genomes from thousands of organisms

How is bioinformatics used following genome sequencing?

Once the genomes have been sequences, large databases are created with biological information and this allows for comparisons and analyses to determine similarities and differences between genomes

List uses of bioinformatics

• Gene therapy

• Evolutionary concepts

• Microbial analysis computing

• Understanding protein structure

• Finding new drugs

• In agriculture, to understand crop patterns, pest control and crop management

Give examples of genetic conditions that can be screened for

• Sickle-cell

• Breast cancer

• Haemophilia

• Cystic fibrosis

• Huntington’s disease

Give benefits of producing human proteins by recombinant DNA technology

• The human version of the protein can be produced instead of another animals

• The rate at which proteins can be produced is much faster than other methods

• Supply is reliable

• The gene product is easily extracted and purified

Why i genetic screening carried out?

To test for and diagnose genetic conditions

May be used in personalised medicine to determine the best treatment for a particular genotype

Define the term gene therapy

Treatment of a genetic condition by altering the individual’s DNA

Give examples of vectors that may be used in gene therapy

• Viruses

• Liposomes

• Naked DNA

How can PCR and DNA testing be used in forensic science?

DNA found at the crime scene can be amplified using PCR and tested to see if it matches the DNA of a suspect

Why is genetic engineering used in crops and livestock?

The quality and the yield can be increased by manipulating the genome

Describe how Bt maize is different from regular maize

Bt maise is resistant to certain insects which improves its yield

It expresses the Bt toxin which comes from a species of bacteria. The Bt toxin is selective to certain pests and does not harm other organism

Describe how salmon have been genetically modified

The gene for regulating growth hormone from the salmon and promoter from ocean pout are inserted in a fertilised egg of the Atlantic salmon, the growth hormone is expressed constantly causing the salmon grow faster

How can genetic engineering increase the production of crops?

Crops may be genetically engineered to contain genes for herbicide resistance

Some crops may be engineered to be resistant to insecticides like Bt maize and cotton

Outline the challenges faced when choosing an appropriate vector for gene therapy

• Retroviruses insert their DNA randomly into the host cell and this disrupts other genes and could lead to inactivation of tumour suppressor genes

• Viral vectors are limited in how much can be carried

• Antiviral immunity can prevent viral vectors delivering the DNA to target cells

• Naked DNA may not be efficiently delivered into cells

What are the social and ethical considerations of using gene therapy?

Moral acceptability of gene therapy and how it may be exploited

Often expensive so it may only available to the rich

What are the social and ethical considerations of using genetic testing?

Psychological burdens on the induvial and their familied

When screening unborn fetuses, the method of obtaining fetal DNA can cause miscarriage

If the test for genetic condition is positive or if the fetus is not the desired sex, the parents may choose to abort

It can affect insurance covers