LOCATING A GENE Flashcards
DNA Probe
short single-stranded section of DNA with base sequence complimentary to part of target gene, it is labelled either radioactively or fluorescently
DNA Probe will be made that has bases complimentary to the portion of DNA whose position we want to find
The DNA strands are separated
The DNA strands are mixed with the DNA Probe, if the desired base sequence is present, the DNA Probes will binds to complimentary bases of the desired gene, this is called DNA Hybridisation
The site at which the DNA Probe has attached itself can be distinguished by looking for the radioactive/fluorescent probe
Therefore, DNA Probes have two benefits
1) It tells you if desired gene is present
2) If desired gene is present, it binds to it, therefore telling you where to find the desired gene
How DNA Probes can help against cancer
They can help identify carrier of the cancer gene
They can identify which cancer gene is present
we need to KNOW THE BASE SEQUENCE of the DNA sequence we are investigating (DNA Sequencing/Sanger Sequencing)
Make four test tubes, one containing many single stranded strands of the DNA sequence as well as a mixture of nucleotides as well as a small quantity of the terminator base Adenine (terminator bases do not form hydrogen bonds) as well as a primer that is fluorescently/radioactively labelled as well as DNA Polymerase. In the second tube, put the same ingredients except put a small quantity of the terminator base Thymine instead of Adenine. Do the same for the other two tubes, with terminator Guanine and terminator Cytosine
The DNA strand being investigated will be copied many time using Polymerase Chain Reaction and added to the four tubes, each DNA strand in the four tubes can bind to a normal nucleotide or the terminator nucleotide forming a DNA fragment using DNA Polymerase
Depending on exactly where the terminator nucleotide binds, DNA synthesis will be terminated after only a few nucleotides of after a long fragment of DNA has been synthesis
The primer will tell us where the binding started and the terminator bases will tell us where it ended
We now need o separate the different length fragments of DNA which is done through Gel Electrophoresis
Obviously before attempting to find the base sequence of the gene, we must find the amino acid sequence, and then find DNA base sequence using the method described above.
DNA Primer
Short length of DNA which is single stranded and has a specific base sequence which indicates where replication will start
Why the DNA fragments formed in the tubes have different base sequences
They have different lengths as strand synthesis stops at terminator nucleotide
Gel Electrophoresis
The DNA fragments are placed onto an agar gel and a voltage is applied across it
Each fragment is negatively charged due to the Phosphate group, therefore will try to move towards the anode (+)
The resistance of the gel means that the larger the fragments, the more slowly they move
The DNA fragments on the gel cannot be seen
Therefore sheet of photographic film placed on agar gel for several hours, radioactivity/fluorescence from each fragment shows where it is on the gel, this method is extremely sensitive
In this way DNA fragments of different lengths are separated
In the exam, for Gel Electrophoresis you will write
Current switched on
Fragments move due to electrical attraction
DNA transferred to nylon membrane
Photographic film placed on gel
Film developed, the radioactivity darkens the film
How scientists can use electrophoresis to estimate the NUMBER OF BASE PAIRS in the separated fragments
Compare the distance moved by the fragment with the distance moved by a fragment of known size
Why base pairs are a suitable way of measuring the length of a piece of DNA
DNA is made of base pairs
Each base pair is same length
How the fragments separated by Gel Electrophoresis may be located by the radioactivity of the Primer
Lay gel close to X-ray film
Develop film
Why it is necessary for the Primer to be radioactively/fluorescently labelled
DNA is not visible on the gel, therefore radioactive/fluorescent label shows up on the photographic film
Restriction Mapping
only small DNA fragments can be sequences in the way described above, larger genes must be cut into smaller fragments by Restriction Endonucleases and each fragment sequenced. Therefore a restriction map is simply a diagram of a piece of DNA marked with the locations of sites where it is cut by restriction enzymes. The fragments are then compared with fragments of known size
If in restriction mapping, three fragments/bands were produced, then we know it has two recognition sites
Restriction Mapping is useful because
Can be used to choose restriction enzymes to generate known-sized fragments
It can aid DNA sequencing of long pieces of DNA (this is the most useful use)
How scientists ensure that the enzyme (Restriction Endonucleases) has completely digested the plasmid DNA
Add the size of the fragments together, if they add up to more than the total length of the original DNA, then they know the enzyme has not fully digested the DNA
Automation of DNA sequencing & Restriction mapping
Instead of a radioactively labelled primer, the four types of terminators are labelled with the fluorescent due
DNA synthesis takes place in a single test tube
DNA synthesis is speeded up by using PCR cycles
Electrophoresis is carried out in a single narrow capillary gel
Results are scanned by lasers and interpreted by computers
PCR can be used to provide fragments required in these techniques
Genetic Screening
Use DNA sequencing and PCR to produce DNA Probes that are complimentary to bases of the defective gene
Fix hundreds of different probes in an array on a glass slide
Add a sample of DNA to the array, any complimentary DNA sequences in the investigated DNA will bind to one or more probes
If it binds, then the individual is said to be a carrier of a genetic disease
If a person is informed that he is at a greater risk of cancer through genetic screening, he may choose
To give up smoking Lose weight Eat more healthy Avoid mutagens Regularly check themselves for cancer To undergo gene therapy
Implication of genetic screening
Who decided who should be screened
Who has access to the test results
Would employers refuse insurance cover if they knew a person has a genetic disorder
What are the responsibilities of someone who carries a gene for an inherited disease
Does mankind have a responsibility to maintain genetic diversity
Who decided what is a defect or a disease
Southern Blotting
DNA is cut using restriction enzyme
Electrophoresis separates DNA fragments according to length
DNA made single-stranded
Transfer to positively charged nylon membrane to which the negatively charged DNA stick
Probe is applied which does complimentary base pairing to the DNA fragments in the nylon membrane
Radioactive strand detected on film
Pattern unique to every individual
Southern Blotting is the method used for
GENETIC FINGERPRINTING; the differences in DNA between humans is found in the non-coding DNA, which is repetitive
The probability of two organisms having the same fingerprints is very low; about 1 in 109
To identify restriction fragments containing a particular gene
GENETIC SCREENING
Genetic fingerprinting is used for
Crime investigation
Determine family relationships
Prevent undesirable breeding
Determine relationships between ancient humans
Establish evolutionary relationships with other species
Measure genetic diversity within a population
Why genetic fingerprinting takes long if the sample is small
PCR is required to provide amplification of the DNA
Some plants can be made as a pesticide by incorporating the pesticide into genome of plant to allow plant to produce toxin, this called genetically modifying crops. (PS Don’t get confused, this isn’t Selective Breeding)
Advantages of making Genetically Modified Crops against pests
More effective than other methods Less use of insecticide Prevent spread of disease Economic benefit to farmer Possible cheaper food
Disadvantages of making Genetically Modified Crops against pests
Plasmid may enter another species May sterilise other species Disruption of food chain Poison may harm other organisms Consumer may consider product as unnatural Unknown side-effects unknown Risk to biodiversity Risk to local farmers
FOR doing genetic engineering to clone cells from human embryos
Embryo can develop into any type of tissue
Easier to use embryo cells than to extract cells from a person
Cells would replicate in patient during mitosis
Permanent cure
Likely to be safer than implants from animals as animals may contain ‘new’ viruses
Only body cells implanted, therefore germ line would not be affected
AGAINST doing genetic engineering to clone cells from human embryos
Long term side effects are unknown
Embryo has potential for development to person therefore could be regarded as murder
