1.7 Genetic techniques Flashcards
why is dna extracted
Test a newborn for a genetic disease
Analyse forensic evidence
Study a gene involved in cancer
what are the steps involved in DNA extraction
Collect cell sample (cheek cells can be collect using a swab)
Add lysis solution
Detergent disrupts the cell membrane and nuclear envelope –causing the cell to burst
The proteinase K breaks down the histones to free the DNA
Add concentrated salt solution - makes the cell fragments stick together
Put the solution in a centrifuge - heavy clumps of protein and cellular debris sink to the bottom. DNA remains in the liquid.
Ethanol is added to isolate the DNA – DNA is not soluble in ethanol and comes out of solution.
what is the POLYMERASE CHAIN REACTION and what are its uses
An extremely important laboratory technique used to multiply tiny amounts of DNA very quickly.
Uses:
Rapid pre-natal diagnosis of genetic diseases
Analysing DNA from preserved fossils
Amplifying DNA from crime scenes
what is the process of pcr
Only a small DNA sample is required eg. Small sample of blood, skin saliva or even hair follicles.
Primers are added – primers attach to sites on the DNA strands that are at either end of the segment you want to copy.
Nucleotides are added – the genetic building blocks to make billions of DNA copies.
DNA Polymerase is added to read the DNA code and attach the complementary nucleotides to create DNA copies.
Note: the DNA polymerase is selected to withstand the high heat of the PCR reaction.
Heating and cooling – in a DNA Thermal Cycler the tube is heated and cooled at specific times which is crucial for making the reaction work.
Heated to 95°C – this separates the DNA double helix
Cooled to 50°C – this causes the primers to join complementarily to the DNA strands
Heated to 72°C – activates the DNA polymerase
These steps continue until the desired amount of DNA is produced.
After 30 cycles there are over a billion fragments containing the target sequence.
what are the purpose of the elements of pcr
heating and cooling
primers
free nucleotides
Heat-resistant enzymes:
Heating and cooling: Allows DNA strands to separate periodically, and primers to anneal when cooled.
Primers: Bind complementary to the target sequence in order to initiate replication. Also prevent two strands from reforming.
Free nucleotides: Synthesise the newly replicating strands.
Heat-resistant enzymes: Enzymes are temperature sensitive and since samples are heated to high temps, hence a non-heat resistant enzyme would be denatured.
what are restriction enzymes
Extracting DNA from cells can be achieved by treating the nuclei with chemicals, which break down the membrane, and isolate the DNA from the nuclear proteins.
Restriction enzymes are found in bacteria, which protect them from viruses) can then be used to cut the DNA at specific positions.
enzymes found in bacteria, which protect them from viruses) can then be used to cut the DNA at specific positions.
what is gel electrophoresis
A common lab technique used to separate large molecules (including nucleic acids and proteins) on the basis of size and charge.
how does gel electrophoresis work
DNA samples (which have been cut up using restriction enzymes) are loaded into wells (at the negatively charged electrode).
As DNA is negatively charged (due to the phosphate group), the samples will move toward the positively charged electrode.
The rate of migration through the gel will depend on several factors – including size.
Larger fragments will move more slowly, whereas smaller fragments will move quickly.
DNA markers are often used. These are samples of KNOWN size and allow us to estimate the size of fragments in our unknown samples.
what did the mapping of genomes determine
allowed researchers to determine which genes belonged to certain chromosomes. These advances help us to understand how diseases develop and are inherited, how cells differentiate, how embryos develop, as well as our own evolution.
how many chromosome pairs, nucleotide bases, genes, repetitive DNA and percent of genes
does the human genome have
23 chromosome pairs
3 x 109 nucleotide bases
Approx. 20,000 + genes
Approx 50% repetitive DNA
Genes represent 1.5-2% of genome
what is DNA profiling
Technique which utilises the fact that the DNA sequence is unique and specific to each individual.
If we all need the same proteins (such as haemoglobin) how is it that we have different DNA?
DNA contains sections of DNA that do not code for protein – called non-coding DNA.
This DNA accumulates mutations over time (as the mutations do not disrupt protein production, and hence do not affect phenotype).
There can be as many as half a million base pair differences on each chromosome of different individuals.
Steps taken to produce a DNA profiling:
DNA sample (from blood, saliva, skin, semen etc) is obtained and multiplied using PCR (polymerase chain reaction).
The DNA sample is digested (cut up) using restriction enzymes. This will produce DNA fragments of different size – the length of fragments will differ from individual to individual as they will have different base sequences.
The fragments are then analysed using gel electrophoresis – which will separate the fragments according to size, giving a unique banding pattern.
A sheet of nylon membrane is placed on top of the gel – transferring DNA from the gel to the nylon membrane.
Radioactive probes are added which bind to specific DNA sequences on the fragments.
Sample is exposed to X-ray film.
The dark bands make up the DNA profile.
what are short tandem repeats
Sections of DNA that a repeated between genes on particular chromosomes, are unique to individuals.
ethics of the collection of genetic information
Deter criminals, reduce crime- Expensive (for now)
Save money long term (e.g. in forensics)-Genetic information can be used against the individual e.g. Insurance companies, employers
Can be used in paternity cases-Lack of consent for infants
Greater understanding of human development-Privacy
Treatment of genetic diseases-Potential to alter information (eugenics)
ETHICS OF GENETIC MANIPULATION
Genetically modified foods can reduce the need for toxic fertilizers and can produce higher, better quality yields
-Loss of variation; possible harmful effects on the ecosystem
Economic profit-Against the laws of nature; environmental and social costs
Treatment of genetic diseases-Expenses associated with biotechnologies – could it create an unfair system whereby only the rich can afford leading to division of class in society?
DNA profiles can be used in paternity cases and in criminal cases-Genetic information can be used against the individual e.g. Insurance companies etc
what is dna sequencing
a laboratory technique used to determine the exact order of nucleotides (bases) in a DNA molecule, which encodes the biological information cells use to develop and operate.
