IB Categories Archives: Topic 3: Genetics
3.4 – Genetic Engineering and Biotechnology
3.4.1 – Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA
This process is also called DNA amplification, and is used to produce enough DNA for procedures such as:
- DNA sequencing
- DNA profiling
- Diagnose disease
- Identify bacteria
It produces more DNA when there is only a small sample available, such as from a crime scene or a long-extinct organism. The cycle of replication happens at an exponential rate, and makes billions of copies in a few hours
Sample obtained (target DNA). DNA denatured by heating at 95°C for 5mins then cooled to 60°C. Primers are bonded (annealed) to each strand. Primers are short strands of DNA which provide a starting sequence for DNA extension. Free nucleotides and DNA polymerase are added. Polymerase binds to primers and synthesises complementary strands of DNA with the free nucleotides, resulting in two copies of the DNA. The process is repeated about 25 times
The Thermal Cycler
Samples in tiny PCR tubes are put in the loading tray and the lid is closed
Machine has heating and refrigeration mechanisms to rapidly change the temperature
Pipettes with disposable tips are used to dispense DNA samples into the PCR tubes
Amount of DNA sample is determined by placing a known volume in the quantitation machine. Often a minimum amount of DNA is required
Control panel allows a number of different PCR programmes to be stored in the machine’s memory. Usually only one is started to run a PCR
3.4.2 – State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size
Gel electrophoresis is a method that separates large molecules, including nucleic acids or proteins based on size, electric charge, and other physical properties. DNA has a slight negative charge due to the phosphates on the backbone, repelled by the negative electrode. Shorter DNA molecules travel further, whilst larger molecules are subject to more friction against the gel. Once stained, the separated molecules in each lane can be seen as a series of bands
- Tray is prepared to hold the gel matrix
- Gel comb is placed in the tray to create wells in the gel
- Agarose gel powder is mixed with a buffer solution. This is heated until dissolved, then poured into the tray to cool. The buffer is a liquid used to carry the DNA in a stable form
- Gel tray is placed in the electrophoresis chamber, which is then filled with buffer to cover the gel. This allows the electric current from the electrodes at either end to flow through it
- DNA samples are mixed with a loading dye to make the DNA visible. This contains glycerol or sucrose to make the DNA heavy so that it sinks to the bottom of the well
- A safety cover is placed over the gel, electrodes are attached to power supply and turned on
- Once the dye marker has moved through the gel, the current is turned off and the gel is removed from the tray
- DNA molecules are made visible by staining the gel with methylene blue or ethidium bromide, which binds to DNA and fluoresces in UV light
- The gel matrix acts as a sieve for the negatively charged DNA molecules as they move towards the positive terminal
- Large molecules have difficulty getting through the holes
- Small molecules move easily
Chromosomes have simple, repetitive sequences of non-coding DNA that are found scattered throughout the genome. Short sequences are called microsatellites or short tandem repeats, Sequence lengths vary considerably between people in the numbers of the repeating unit
Gel electrophoresis is used in DNA fingerprinting, which is a form of DNA profiling, used to differentiate one individual from another
The technique is used in forensic crime investigations, parentage issues, animal breeding pedigrees and disease detection.
- DNA is extracted
- Microsatellites are amplified using PCR
- Specific primers for microsatellites are used
- Fragments separated in a gel electrophoresis
3.4.4 – Describe the application of DNA profiling to determine paternity and also influence in forensic investigations
DNA samples are taken from mother, child and potential fathers. All the DNA fragments from the child must match with either the mother or father. The band on the child’s fragments are either found on the mother or the father (Male 1)
3.4.5 – Analyse DNA profiles to draw conclusions about paternity or forensic investigations
See above statement
3.4.6 – Outline three outcomes of the sequencing of the complete human genome
The Human Genome Project was an international research effort to identify and map all the human genes. We have benefitted greatly from the completion of this project, including:
- A knowledge of the number of human genes
- The location of specific genes. There are approximately 30,000 genes in the human genome, which have all been identified.
- All the information has been stored in a database for future reference and research.
- Allowed for the discovery of proteins and their specific functions.
- The technologies that were developed for this research also have uses in other areas.
- The evolutionary relationships between organisms can be identified using genetics.
- However, there are ethical, legal and social issues that have arisen from the project.
3.4.7 – State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal
The genetic code is universal, with all known organisms using the same nucleic acids to code for proteins. In principle, if we transfer a gene from one species to another, it should be transcribed and translated into the same protein.
3.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase
In prokaryotic organisms, most of the DNA is in one circular chromosome. They also have plasmids, which are smaller circles of DNA floating freely in the cytoplasm. Plasmids can be removed and cleaved by restriction enzymes at target sequences.
DNA fragments from another organism can also be cleaved by the same restriction enzyme. Pieces can be added to the open plasmid and spliced together by ligase. Recombinant plasmids can be inserted into new host cells and then cloned.
The plasmid is cut using restriction enzymes, leaving sticky ends, or exposed nucleotide bases.
The DNA containing the desired gene will have been isolated using gel electrophoresis. This DNA strand will also be cut at s specific recognition site using the same restriction enzymes. This produces a fragment with complementary sticky ends to those on the plasmid.
The plasmids and the DNA fragments are mixed together in the presence of the enzyme ligase. The hydrogen bonds form using complementary base pairing and the sugar-phosphate backbone is joined through annealing.
3.4.9 – State two examples of the current uses of genetically modified crops or animals
Added Retinol in Rice
Retinol, or Vitamin A1, is essential for the development of an effective immune system, normal vision and growth. Deficiency leads to stunted growth, weakened immune system, loss of night vision and possible blindness. In third world countries, deficiency is coupled with malnutrition and disease. Children are also more likely to die from disease when they are vitamin A deficient.
Beta-carotene is used by the body to make retinol. Normal rice does not contain retinol or beta-carotene, but it does contain a molecule that is normally used to make beta-carotene. However, the gene and enzymes to manufacture retinol are missing.
GM rice contains the gene for the manufacture of beta-carotene. The gene was sourced from Erwinia bacterium or the common daffodil. Transgenic rice is usually yellow due to the presence of beta-carotene, which is crossed with local strains of rice. As a result, communities are able to have more nutrients in their diet.
Herbicide Resistance in Crop Plants
Weeds use soil nutrients that crops need to grow and the competition reduces productivity and efficiency of farming. Herbicides are used to kill weeds. They must be used before planting, as they also kill crops.
Some crops have been made resistant to major herbicides because they can produce an enzyme that breaks down glyphosate, found in a major herbicide. Herbicides can be used after planting to kill weeds.
3.4.10 – Discuss the potential benefits and possible harmful effects of one example of genetic modification
3.4.11 – Define clone
A group of genetically identical organisms or a group of cells derived from a single parent cell
3.4.12 – Outline a technique for cloning using differentiated animal cells
Donor somatic cells were taken from the udder of the original sheep that was to be cloned and
cultured in a low-nutrient medium to stop division of the cell, making it dormant. An unfertilised
ovum was taken from a ewe and stimulated to superovulate by hormone FSH, producing a large
number of ova. The nucleus, containing DNA, was removed from the ovum using micromanipulation techniques. The ovum was left with no nucleus, but still had a cytoplasm and cellular machinery to produce an embryo.
The dormant donor cell and egg cell were fused using a gentle electrical pulse. The ovum retained its ability to replicate chromosomes and divide by mitosis. Cell division was triggered and when it reached the 16-cell stage, it was implanted into a surrogate mother sheep. Dolly, the cloned sheep, was genetically identical to the donor sheep. However, they experienced a different set of environmental conditions.
3.4.13 – Discuss the ethical issues of therapeutic cloning in humans
3.3 – Theoretical Genetics
3.3.1 – Define genotype, phenotype, dominant allele, recessive allele, codominant alleles, locus, homozygous, heterozygous, carrier and test cross
Genotype – The alleles of an organism Phenotype – The characteristics of an organism
Phenotype – The characteristics of an organism Dominant Allele – An allele that has the same effect on the phenotype whether it is present
Dominant Allele – An allele that has the same effect on the phenotype whether it is present in the homozygous or heterozygous state Recessive Allele – An allele that only has an effect on the phenotype when present in the
Recessive Allele – An allele that only has an effect on the phenotype when present in the homozygous state
Codominant Alleles – Pairs of alleles that both affect the phenotype when present in a heterozygote
Locus – The particular position on homologous chromosomes of a gene
Homozygous – Having two identical alleles of a gene
Heterozygous – Having two different alleles of a gene
Carrier – An individual that has one copy of a recessive allele that causes a genetic disease in individuals that are homozygous for this allele
Test Cross – Testing a suspected heterozygote by crossing it with a known homozygous recessive
3.3.2 – Determine the genotypes and phenotypes of the offspring of a monohybrid cross using a Punnett grid
Genetics crosses allow us to determine the genotype and phenotype of offspring, using the Punnett grid method.
Cross = Heterozygote (Rr) x Heterozygote (Rr)
In this example, there are three genotypes in the ratio:
1RR : 2Rr : 1rr
The phenotypes for the offspring with the dominant gene will all express this gene in their phenotype. Only the genotype rr results in a phenotype with the recessive gene. Hence, there are two phenotypes in the ratio:
3 Dominant : 1 Recessive
3.3.3 – State that some genes have more than two alleles (multiple alleles)
There are some genes that have more than two alleles. However, an individual can only possess two alleles. Multiple alleles increase the number of different phenotypes in a given population. Multiple alleles can be dominant, codominant or recessive.
3.3.4 – Describe ABO blood groups as an example of codominance and multiple alleles
In the human ABO blood group system, the blood group can be determined by three different alleles, meaning that there a multiple alleles. These are A, B and O. ABO antigens are sugars attached to the surface of red blood cells. Each allele codes for enzymes that join theses sugars together.
O produces a non-functioning enzyme that does not make any changes to the basic molecule. A and B are codominant (expressed equally), producing different antigens. These antigens react with antibodies present in the blood from other people, which must be matched for transfusion.
These can also be written IA, IB and i. I stand for immunoglobin
Gender is determined by the sex chromosome inherited from each parent. Males are referred to as the heterogametic sex. Each somatic cell has one X chromosome and one Y chromosome. Females are homogametic. Somatic cells with two X chromosomes. X chromosomes are longer than Y chromosomes. Sex chromosomes are the 23rd pair.
The only possible phenotypes from a cross are male and female – XX or XY. The ratio of karyotypes is therefore:
1XX : 1XY
3.3.6 – State that some genes are present on the X chromosome and absent from the shorter Y chromosome in humans
There is a size difference between the X and Y chromosomes. A male will only have one allele for genes that occur in the non-homologous region of the X chromosome. Genes in the homologous region will have to alleles, functioning like other genes. Females always have two alleles because the complete length of X chromosome has a homologous pair.
3.3.7 – Define sex linkage
Genes carried on only one of the sex chromosomes and which therefore show a different pattern of inheritance in crosses where the male carries the gene from where the female carries the gene
When a gene is sex linked, the characteristic is usually seen in the heterogametic sex (human males). Examples include red-green colour blindness and haemophilia.
3.3.8 – Describe the inheritance of color blindness and hemophilia as example of sex linkage
Red-Green colour blindness is produced by a sex-linked, recessive allele. The gene loci is on the non-homologous region of the X chromosome. Males must inherit the gene from their mothers. Males cannot pass the gene onto their sons. A female can carry the gene without expressing it.
Haemophilia is produced by sex-linked, recessive alleles. Males are always affected, but females can be carrier. It is inherited from the mother. Haemophilia is a condition in which blood clotting factor cannot be produced, causing uncontrolled bleeding. It is more common in men than women.
3.3.9 – State that a human female can be homozygous or heterozygous with respect to sex-linked genes
Homozygous – having two identical alleles of a gene
Heterozygous – having two different alleles of a gene
Females can be homozygous for sex-linked alleles, with both alleles have the same gene. On the other hand, they can be heterozygous, with each chromosome carries a different gene.
3.3.10 – Explain that female carriers are heterozygous for X-linked recessive alleles
Carriers for recessive alleles have both the dominant and the recessive allele. The disease is recessive, so it is not expressed in the carrier’s phenotype. They have two different alleles of a gene.
3.3.11 – Predict the genotypic and phenotypic ratios of offspring and monohybrid crosses involving any of the above pattern of inheritance
Cystic fibrosis is found on the autosomal chromosomes. It is a recessive disorder in which the allele key is dominant CF and Cf for the recessive allele.
Therefore the ratio of phenotypes is:
3 No Disease : 1 With Disease
Therefore, the child has a 75% chance of not having the disease.
3.3.12 – Deduce the genotypes and phenotypes of individuals in pedigree charts
Geneticists collect information about individuals and relatives within a family. They construct diagrams of inheritance, or family trees, which are called pedigrees. Circles are females, squares are males, diamonds are for unknown, small black circles means died at infancy. Carriers are marked with a small black dot in the centre. Identical and fraternal twins are shown as:
Black means the individual has the condition, white means unaffected. Mating is indicated by a horizontal line
For dominant and recessive alleles, upper case and lower case letter should be used. Letter representing alleles should be chosen with care to avoid confusion between the cases. For co-dominance, the main letter should relate to the gene, and the suffix to the allele
i.e. Sickle cell anaemia: HbA and Hbsb
Individuals are identified by their generation and order number. Generations are written I, II, III, IV, etc. Orders are simply 1,2,3. The propositus is the person through whom the pedigree is discovered.
3.2 – Meiosis
3.2.1 – State that meiosis is a reduction division of a diploid nucleus to form haploid nuclei
Meiosis consists of two divisions. In the first division, the diploid cell replicates its chromosomes then divides, forming two diploid cells. However, in the second division, there is no DNA replication, so the resultant cells only contain half the DNA. Therefore, each diploid cell that undergoes meiosis will produce four haploid cells.
Diploid cells contain two sets of chromosomes, whilst haploid cells only contain one set.
Chromosomes in a diploid cell which contain the same sequence of genes, but are derived from different parents. During meiosis, the homologous chromosomes will pair up.
They share the same structural characteristics, such as length, shape and the loci of their genes. However, they may have different alleles of each gene.
3.2.3 – Outline the process of meiosis, including pairing of homologous chromosomes and crossing over, followed by two divisions, which result in four haploid cells
In this stage, the nuclear membrane is intact and the chromosomes are not densely
wound. In the G1 Stage, the chromosomes are still single DNA molecules with their
histones. In the S1 Stage, there is replication of chromosomes. Sister chromatids are held together at the centromere.
The DNA condenses by super coiling and become visible. Homologous chromosomes
pair up. Crossing over occurs as there is breakage and reunion of parts of chromatids
– this is the exchange of genetic material between the non-sister chromatids. The nuclear membrane breaks down and spindles form from the microtubules at opposite ends of cell, organised by the centrioles
The pairs of homologous chromosomes line up along the equator. The spindle fibres attach to the centromere. The random orientation of the chromosomes means that the maternal or paternal chromosome may move to either pole.
Spindle fibres shorten, pulling the chromosomes towards the opposite poles. Sister chromatids remain attached at the centromere. Each pole will have a complete haploid set of chromosomes consisting of one member of each homologous pair.
The spindle breaks down and the nuclear membrane reforms. Each daughter nucleus contains two sister chromatids for each chromosome, attached at the centromere. Crossing over means that the two sister chromatids are not identical. The cell divides and the two resulting cells are haploid cells
The nuclear membrane breaks down and a new spindle forms. The chromosomes appear as two chromatids joined at the centromere.
The chromatids arrange at the equator and the spindle fibres bind to both sides of the centromeres
The spindle fibres contract, causing the centromeres divide. Sister chromatids move to opposite poles.
Spindle breaks down and nuclear envelopes reform around the sets of daughter chromosomes. The cells divide again through cytokinesis, resulting in four haploid cells.
Meiosis contributes to genetic variability as it reduces chromosomes by half, permitting fertilisation and combination of genes from two parents. There is random assortment of maternal and paternal chromosomes during meiosis I, meaning that the genes from either parent have an equal chance of entering a cell. There is also recombination of segments of individual paternal and maternal homologous chromosomes due to crossing over.
3.2.4 – Explain that non-disjunction can lead to changes in chromosome number, illustrated by reference to Down syndrome
Non-disjunction is the term for the failure of a pair of chromatids to separate and go to opposite poles during the division of the nucleus. In meiosis, this results in gametes with more than and less than the haploid number of chromosomes.
A pair of number 21 chromosomes fails to separate during the formation of an egg or sperm, called nondisjunction, or trisomy 21. When the egg is fertilised to form an embryo, three copies of chromosome 21 are present, which is copied to every cell in the baby’s body. The risk of nondisjunction increases as women get older.
3.2.5 – State that, in karyotyping, chromosomes are arranged in pairs according to their size and structure
Karyotype – the chromosome complement of a cell or whole organism
Karyotypes show the number, size and shape of chromosomes during metaphase of mitosis, They are prepared from the nuclei of cultured white blood cells. Scanning electron micrographs take a picture of the chromosomes. Chromosomes are arranged into pairs on the basis of length, pattern of banding and shape, and will appear as pairs of sister chromatids.
This shows a male human (Y) with no visible chromosomal abnormality
3.2.6 – State that karyotyping is performed using cells collected by chorionic villus sampling or amniocentesis, for pre-natal diagnosis of chromosome abnormalities
Chorionic Villus sampling
A sample is taken from the fetal tissue part-buried in the wall of the uterus in the period 8 10 weeks. The purpose is to diagnose any conditions or diseases in the unborn child. These cells have the same genotype as the embryo. A sample is taken from the chorion, an extra embryonic membrane. A catheter tube is inserted via the vagina and a sample is taken. The sample is cultured to produce cells for karyotyping
Remove , produced by the amnion membrane and contains cells . The cells are removed by inserting a needle into the abdominal wall, myometrium and into the amniotic fluid. The fluid is centrifuged, cells are incubated and then karyotyped. The fluid (supernatant) can be used to test for neural tube disorder such as spina bifida.
Preparation of the Karyotype
A sample is taken. The cells are centrifuged and treated to make the cells swell up and chromosomes spread out. White blood cells are treated with a drug that causes them to go into mitosis, then one to halt the process at metaphase. Cell suspension is placed on a slide, dried and stained to show the banding. A photograph of chromosomes is taken and is cut up so that the chromosomes can be arranged in homologous pairs
Karyotyping can be carried out when chromosomes from the metaphase are available. Appropriate staining techniques are used to reveal characteristic banding patterns. The number of
chromosomes is counted. The chromosomes are then organised into pairs based on length, position of the centromeres, banding patterns and the satellite ends.
3.1 – Chromosomes, Genes, Alleles and Mutations
Chromosomes are composed of two daughter chromatids which are joined at the centromere. Chromosomes are mainly comprised of DNA and histone proteins
3.1.2 – Define gene, allele, and genome
Allele – An allele is a specific form of a gene, differing for other alleles by one or a few bases
only. They occupy the same gene locus as the other alleles on the gene Genome – The whole of the genetic information of an organism
Genome – The whole of the genetic information of an organism 4.1.3 – Define gene mutation
3.1.3 – Define gene mutation
A gene mutation is a change in the base sequence of an allele This may produce a different amino acid sequence in the protein translated, which may not
This may produce a different amino acid sequence in the protein translated, which may not be beneficial. A substance that causes mutation is called a mutagen, including radiation and chemicals.
Deletion is when one of the bases is removed, changing the whole gene. Insertion involves the addition of a base, which also changes the whole gene. Substitution is when a base is changed, altering only one amino acid. However, this will still affect the shape of the protein.
3.1.4 – Explain the consequence of a base substitution mutation in relation to the processes of transcription and translation, using the example of sickle-cell anaemia
Sickle cell anaemia is a genetic disease. It has a frequency of about 1 in 655 African Americans. The condition is inherited, and cannot be contracted by infectious routes. The affected gene is found on chromosome 11. The sequence that codes for the sixth amino acid normally has the base sequence GAG, which codes for glutamic acid. This amino acid carries a negative charge. However, the substitution produces a different sequence, GUG, which codes for the neutral amino acid valine. The result is that the beta chain changes shape.
Haemoglobin is made up of four proteins, two of which can affected by the mutation. The usual shape of the red blood cells is a biconcave disc. However, when there is mutation, the cells become shaped like a sickle. As a result, the red blood cells cannot carry oxygen, causing anaemia. Furthermore, the irregular shape of the cells means that they do not move through the bloodstream properly, causing blockages in places such as the kidney tubules. This may damage the kidney and possibly lead to death.
In areas where malaria is common, those with the sickle cell anaemia trait are resistant to the infection. This is because normal blood cells are affected by the disease. As a result, those who do not have the mutation are more likely to die from malaria. In these regions, sickle cell anaemia has become more common since it gives carriers an advantage.