Recombinant DNA technology - application

Question 1

What are DNA probes?

Answer 1

A short, single stranded length of DNA with a label attached to make it easily identifiable.
Radioactively labelled probes are made of nucleotides with the isotope P32, and identified using an X-ray film exposed to radioactivity.
Fluorescently labelled probes emit light under certain conditions, e.g. when the probe has bound to the DNA sequence.

Question 2

How do DNA probes identify particular alleles of genes?

Answer 2

A DNA probe is made that has complementary base sequences to the DNA of the allele.
The double stranded DNA being tested is treated to separate its strands.
The strands are mixed with the probe, which binds to the complementary base sequence of one of the strands - DNA hybridisation.
The site it binds to can be identified.

Question 3

What is DNA hybridisation?

Answer 3

Used to sequence the exact order of bases in a length of DNA of an allele trying to be located, so a specific probe can be made.
It takes place when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.

Question 4

What happens before hybridisation?

Answer 4

The two strands of DNA must be separated.
This is done by heating DNA until its double strand separates into complementary single strands (denaturation).
When cooled, the complementary bases recombine (anneal) with each other to reform the original double strand.

Question 5

What are the implications of hybridisation?

Answer 5

Given sufficient time, all strands in a mixture of DNA will pair up with their partners.
If however, other complementary sections of DNA are present in the mixture as the DNA cools, these are just as likely to anneal with one of the separated DNA strands as the two strands are with one another.

Question 6

What is location of alleles?

Answer 6

Using DNA probes and DNA hybridisation, it is possible to locate a specific allele of a gene.
This could be used to determine whether someone possesses a mutant allele that causes a particular genetic disorder.

Question 7

What is the process of locating specific alleles of genes?

Answer 7

The sequence of nucleotide bases of the mutant allele is determined, using DNA sequencing techniques, or by referring to the genetic library of bases sequences of genetic diseases.
A fragment of DNA is produced with complementary base sequences to the mutant allele.
Multiple copies of the DNA probe are formed using PCR.
A DNA probe is made by attaching a marker to the DNA fragment.

Question 8

What is the process of locating specific alleles of genes - hybridisation onwards ?

Answer 8

DNA from the suspected person is heated to separate the strands.
The strands are cooled in a mixture containing many DNA probes.
If the DNA contains the mutant allele, one of the probes is likely to bind, because the base sequences are complementary.
The DNA is washed clean of any unattached probes.
The remaining hybridised DNA is fluorescently labelled with the dye attached to the probe.
The dye is detected by shining light onto the fragments causing the dye to fluoresce, detected by a microscope.

Question 9

What are the origins of genetic disorders?

Answer 9

Many genetic disorders are the result of gene mutations.
If the mutations results in a dominant allele, all individuals will have the disorder, if recessive, then only homozygous individuals will.
Heterozygous individuals will be carriers of the disease, and will not display symptoms, but are capable of passing it on to offspring.

Question 10

Why is it important to genetic screen?

Answer 10

Individuals who may carry a mutant allele often have a family history of a disease.
Screening can determine the probabilities of a couple having offspring with a genetic disorder.
So potential parents at risk can obtain advice from a genetic counsellor about the implications of having children.

Question 11

What is the process of genetic screening?

Answer 11

It is possible to fix hundreds of different DNA probes in a pattern on a glass slide.
By adding a DNA sample to this array, any complementary DNA sequences in the donor will bind to one or more probes.
It is therefore possible to test simultaneously many genetic disorders by detecting fluorescence that occurs where binding has happened.

Question 12

What is another use of genetic screening?

Answer 12

The detection of oncogenes, which cause cancer.
Cancers may develop from mutations that prevent tumour suppressor genes inhibiting cell division.
Mutations on both alleles must be present to inactivate a tumour supressor gene and to develop a tumour.
Some people inherit a mutated tumour suppressor gene, and so are at greater risk of developing cancer.

Question 13

What is the result of genetic screening for cancer?

Answer 13

Individuals more at risk of cancer can make informed decisions about their lifestyle and future treatment.
They can choose to give up smoking, lose weight, eat healthier and avoid mutagens.
They can check themselves more regularly for signs of cancer, which can lead to earlier diagnosis and better chance of successful treatment.

Question 14

What is the use of screening for personalised medicine?

Answer 14

Genetic screening allows doctors to provide advice and health care based on an individual’s genotype.
An individual’s genes can mean that a particular drug may be either more or less effective in treating a condition.

Question 15

What is personalised medicine?

Answer 15

Genetic screening can determine more exactly the dose of a drug to produce the desired outcome.
This can save money, that would otherwise be wasted on overprescribing drugs.
It can also avoid medications that could cause harm or avoid raising false hopes.

Question 16

What is personalisation of prescribing painkillers?

Answer 16

To function effectively, many pain medications need a specific enzyme to activate them.
About half the population have this enzyme.
Screening for the presence of these genes allow the dosage to be adjusted to compensate for how the genes affect the individual’s metabolism of the painkiller.

Question 17

What is the personalisation of vitamin E?

Answer 17

Among people who have diabetes, vitamin E reduces the risk of cardiovascular disease for those with certain genotypes, but increases for those with a different genotype.
It is advantageous to screen before advising on taking vitamin E supplements.

Question 18

What is genetic counselling?

Answer 18

A form of social work, where advice and information are given that enable people to make personal decisions about themselves or offspring.
One aspect is to research family history of an inherited disease.

Question 19

What is the use of genetic counselling?

Answer 19

A counsellor can inform an individual about the emotional, psychological, medical, social and economic consequences of having offspring with a genetic disease.
The couple can then decide whether or not to have children.
It can make them aware of further medical tests to give a more accurate prediction, e.g. IVF with screening of embryos.

Question 20

What can genetic screening provide a genetic counsellor with?

Answer 20

A basis for informed discussion, and in the case of cancer, screening can help detect:
Oncogene mutations, which can determine the type of cancer and so the most effective drug or radiotherapy.
Gene changes and how different treatments effect, e.g. herceptin is most effective for certain types of breast cancer.
A single cancer cell among millions of normal cells, thus who is at risk of relapse from leukaemia.

Question 21

What is genetic fingerprinting?

Answer 21

A diagnostic tool used widely in forensic science, plant and animal breeding, and medical diagnosis.
It is based on the fact that every individual except identical twins, have unique DNA.

Question 22

What does genetic fingerprinting rely on?

Answer 22

The genome of most eukaryotes contains many repetitive, non-coding bases of DNA, but may still be functional.
Non coding bases are Variable Number Tandem Repeats (VNTRs).
Each individual has a unique pattern of number and lengths of VNTRs.
The more closely related an individual, the more similiar the VNTRs.

Question 23

What is gel electrophoresis?

Answer 23

It separates DNA fragments according to their size.
The fragments are placed on to an agar gel and a voltage is applied across is.
The resistance of the gel means that the larger the fragments, the slower they move.
Only DNA up to 500 bases can be sequenced, larger genes must be cut into smaller fragments by restriction endonucleases.

Question 24

What is gel electrophoresis with labels?

Answer 24

If the fragments are labelled, e.g. radioactive probes, their final positions in the gel can be determined by placing a sheet of X-ray film over the agar gel for several hours.
The radioactivity from each fragment exposes the film.

Question 25

What is the stages of making a genetic fingerprint?

Answer 25

Extraction, digestion, separation, hybridisation and development.

Question 26

What is extraction?

Answer 26

Even the tiniest sample of animal tissue - blood or hair root, is enough to give a fingerprint.
The DNA is extracted by separating it from the rest of the cell.
The amount of DNA can then be increased using PCR.

Question 27

What is digestion?

Answer 27

The DNA is cut into fragments, using the same restriction endonucleases.
The endonucleases are chosen for their ability to cut close to, but not within, the target DNA.

Question 28

What is separation?

Answer 28

The fragments of DNA are separated according to size by gel electrophoresis by electrical voltage.
The gel is then immersed in an alkali in order to separate the double strands.

Question 29

What is hybridisation in fingerprinting?

Answer 29

Radioactive DNA probes are used to bind with VNTRs.
The probes have complementary bases sequences to VNTRs, and bind under specific conditions - pH and temperature.
The process is carried out with different probes, which bind to different sequences.

Question 30

What is development in fingerprinting?

Answer 30

An X-ray film is put over the nylon membrane.
The film is exposed by the radiation from the radioactive probes.
Because these points correspond to the position of the DNA fragments as separated during electrophoresis, a series of bars is revealed.
The pattern of bands is unique to every individual.

Question 31

What is the interpretation of genetic fingerprinting results?

Answer 31

DNA fingerprints from 2 samples are visually checked, e.g. blood at a crime scene and from a suspect.
If there seems to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machine, which calculates the length of the DNA fragments from the bands.
It does this by measuring the distances travelled during electrophoresis by known lengths of DNA.
The odds are calculated of someone else having an identical fingerprint.
The closer the match between the patterns, the greater the probability of the 2 sets being from the same person.

Question 32

What is the use of DNA fingerprinting in genetic relationships?

Answer 32

It can resolve questions of paternity.
Individuals inherit half their genes from their mother, and half from the father.
So each band on a DNA fingerprint should have a corresponding band on one of the parents fingerprint.

Question 33

What is the use of DNA fingerprinting in genetic variability?

Answer 33

The more closely individuals are related the closer the resemblance of their genetic fingerprints.
A population whose members have similar genetic fingerprints has little genetic diversity.

Question 34

What is the use of DNA fingerprinting in forensic science?

Answer 34

DNA is often left at a crime scene - blood, semen and hair.
Genetic fingerprinting can establish whether a person is likely to have been present at the crime scene, although it does not actually prove that they carried out the crime.

Question 35

How does DNA fingerprinting in forensic science not prove someone committed a crime?

Answer 35

The DNA may have been left on some other, innocent occasion.
The DNA may belong to a very close relative.
The DNA sample may have been contaminated after the crime, either by the suspect’s DNA or chemicals that affected the action of the restriction endonucleases.

Question 36

What is the use of probability in forensic science?

Answer 36

The probability that someone else’s DNA might match that of the suspect has to be calculated.
This is based on the assumption that the DNA which produces the banding patterns is randomly distributed in the community.
This might not always be the case, e.g. in religious or ethinic groups they have partners from within their own small community.

Question 37

What is Hungtington’s disease?

Answer 37

Hungtington’s results from a 3 base sequence AGC at one end of a gene on chromosome 4 being repeated over and over again.
People with less than 30 repeats are unlikely to get it, while over 38 repeats are almost certain.
If they have over 50 repeats, the onset will occur earlier than average.

Question 38

What is the medical diagnosis of Hungtington’s disease?

Answer 38

A sample of DNA from a person with the allele for Hungtington’s disease can be cut with restriction endonucleases and a DNA fingerprint prepared.
This can then be matched with fingerprints of people with various forms of the disease and those without the disease.
The probability of developing the symptoms can then be determined.

Question 39

What is the use of DNA fingerprints in plant and animal breeding?

Answer 39

Fingerprinting can be used to prevent undesirable inbreeding during breeding programmes on farms or zoos.
It can also identify plants or animals with a particular allele of a desired gene.
Individuals can be selected for breeding to increase the probability of their offspring having the characteristic that it produces.