Define Gel Electrophoresis
A technique used to seperate DNA fragments according to size, used in DNA profiling to give a unique pattern/sequence
Define Polymerase Chain Reaction
A technique used to amplify small quantities of DNA of a specific DNA region
What is Restriction Enzyme Digestion?
Cuts the DNA at specific sequence / site
What is Profiling?
Provides a unique pattern of bands from a DNA sample , used to compare samples
What is DNA sequencing?
DNA sequencing is the process of determining the sequence of nucleotides e.g. AATTT
What is DNA extraction?
Process by which DNA is removed from the nucleus and from the cells
What are recognition sites?
An enzyme will always cut the DNA at a point where there is a specific sequence of bases
What does DNA ligase do?
An enzyme found in E.coli bacteria that is used to ‘glue’ together short strands of DNA during replication
What are plasmids?
Small circular strand of DNA distinct from the main bacterial genome, it is composed of only a few genes and is able to replicate independently within a cell
What does ‘Ligase’ refer to?
An enzyme capable of combining two small components of single strand DNA into one single structure
What are the steps in Polymerase Chain Reaction?
- DNA is denatured at 94-96 degrees, a double strand is separated into 2 single strands
- Annealing primers activate between 50-60 degrees, temperature drops slightly and then primers attach/anneal and bind to each strand and their complementary bases, a new DNA is then synthesised
- At 72 degrees Taq polymerase attaches to the primer and extends/syntheses the new DNA strand
What is the application of PCR?
- Detecting hereditary diseases
- Detection of viral diseases
- Forensic science - when needing to amplify small amounts of blood, semen, hair etc
Explain the process of Gel Electrophoresis
- Make the gel
- Add comb to the gel and allow it to set
- Add dye to the DNA sample
- Place agarose gel into the tank - electrophoresis
- The buffer (liquid) is added to cover the gel
- DNA sample is loaded into the wells of the gel
- An electric current is applied
- DNA is negatively charged and therefore the DNA fragments move from the negative end to the positive end as the DNA is attracted to the positive end
- Smaller DNA segments move faster and farther than larger DNA fragments
- Gel is stained with DNA binding dye for a few minutes
- Gel is placed under UV light and the sequence will fluoresce, showing the band sequence
Explain the process of DNA sequencing
- The mixture is first heated to denature the template DNA (separate the strands)
- The temperature is then lowered slightly so that the primer can attach/enter
- Temperature is then slightly raised and the enzyme binds to the DNA and elongation occurs
- DNA will elongate until a dideoxynucelotide binds then the sequence wills top as no more can bind as the dideoxynucelotide lacks OH, this results in the collection of DNA strands in different sizes and lengths
- The DNA then goes through a polyacymide gel which s more porous in a DNA sequencer
- The DNA sequencer then analyses the gel and the fragments migrate according to size and each is detected as it passes through a laser beam light, each dideoxynucelotide emits a different colour and is recorded as a colour band on a stimulated gel image which gives us the exact DNA sequence
What are gene probes?
Single stranded DNA labelled with a fluorescent marker to detect the presence of specific DNA sequences on another DNA or RNA molecule. It can be sued to detect hereditary diseases such as cystic fibrous or Huntington’s disease. To detect the disease the probe is made to specifically bind to the specific DNA sequence of the disease
What is gene therapy?
Aim to treat or cure genetic abnormalities by replacing faulty genes with healthy ones. The genes can be used as the treatment. Gene therapy is taking place with single gene disorders such as cystic fibrous and Huntington’s disease. Researchers believe gene therapy may slow down or prevent its development. They have been able to silence the gene
What is cystic fibrous?
A life threatening genetic disorder which may shorten life expectancy. It affects the lungs, pancreas, liver and reproductive organs. It involves the secretion of a sticky mucus by mucus glands. In the lungs it clogs up the air passages,traps bacteria leading to bacterial infection susceptibility. In the pancreas it prevents secretion of pancreatic enzymes for digestion leading to problems with digestion.
What is recombinant DNA?
- Involves isolating the DNA and amplifying it and insertion of gene/DNA segment into transgenic bacteria
- Can make genes in one organism (bacteria) and place them into another organism (humans)
How has treatment for diabetes been influenced by Recombinant DNA
Diabetes treatment (insulin) was initially derived from the pancreas of pigs an cattle however it was impure and variable strength that have could possibly been contaminated resulting in side effects, however new treatment using the technique of recombinant DNA the human gene that was code for insulin was introduced into bacterial cells manufacturing large quantitatives of insulin that was identical to human insulin eliminating contamination
How has treatment for haemophilia been influenced by Recombinant DNA
Haemophilia affects the blood clotting factor VIII, a person suffering has a small supply of this factor and therefore a person suffering is unable to form blood clots accurately and could die from excess bleeding. In the past factor VIII was obtained by donors however there was a risk of contamination transmission of viral diseases such as HIV and hepatitis C.
What is tissue engineering?
To restore healthy tissues or organs, it eliminates the need for organ/tissue transplants or artificial transplants. It requires abundant cell supply and can be induced to grow on a scaffold of natural or synthetic material to produce a 3D tissue
What are the steps of Recombinant DNA
- The gene of interest is isolated and cut out using a restriction enzyme
- Isolate a plasmid from bacterial cell and cut it out with the same type of restriction enzyme used in step 1
- Splice the human DNA into the plasmid using DNA ligase enzymes to join the sticky ends
- Treat the bacterium so it takes up the recombinant plasmid. Once this is successful the bacterium will multiply so that either the human gene or product of the gene can be used
