Chapter 7 DNA Detective Complex Patterns of Inheritance and DNA Fingerprinting PowerPoint lecture prepared by Steve McCommas Southern Illinois State University
DNA Detective 1918: the Romanovs and four servants were murdered by Communists 1991: shallow grave containing bones of at least nine people dug up Were any of these the Romanovs? If so, which ones?
7.1 Forensic Science Bones seemed to belong to six adults and three children Sexing was inconclusive, due to decomposition of pelvises Skeletons might be the Romanovs. Could resemblance among relatives be useful?
7.2 Dihybrid Crosses Dihybrid crosses: crosses involving two genes simultaneously Mendel s peas: seed color and seed shape are on different chromosomes. Y = yellow seed color; y = green seed color; R = smooth seeds; r = wrinkled seeds Cross between two double heterozygote parents: YyRr x YyRr The following Punnett square shows expected numbers of genotypes and phenotypes:
7.2 Dihybrid Crosses - Punnett Square RrYy RrYy Possible types of ovules Possible types of pollen RRYY round, yellow RRYy round, yellow RrYY round, yellow RrYy round, yellow 9 3 3 1 Phenotype Genotype Round, yellow RRYY, RrYY, RRYy, RrYy Round, green Rryy, Rryy Wrinkled, yellow rryy, rryy Wrinkled, green rryy RRYy round, yellow RrYY round, yellow RrYy round, yellow Rryy round, green RrYy round, yellow Rryy round, green RrYy round, yellow Rryy round, green rryy rryy wrinkled, yellow wrinkled, yellow rryy wrinkled, yellow Rryy wrinkled, green Figure 7.1
7.2 Dihybrid Crosses The Tsar and Tsarina were both heterozygotes for hair texture and eye color. Due to random alignment of chromosomes and independent assortment, they could form the following gametes:
7.2 Dihybrid Crosses (a) One possible Metaphase I alignment Two types of gametes Tsarina Cc Dd Meiosis Wavy hair Dark eyes (b) Another possible Metaphase I alignment Two other types of gametes Tsarina Cc Dd Meiosis Wavy hair Dark eyes Figure 7.2a b
7.2 Dihybrid Crosses Their gametes could then potentially produce the following offspring: (c) Punnett square for the mating of the Tsar and the Tsarina Tsar ccdd (straight hair, dark eyes) Tsarina CcDd (wavy hair, dark eyes) Possible types of eggs Possible types of sperm cd CcDD Wavy hair Dark eyes CcDd Wavy hair Dark eyes ccdd Straight hair Dark eyes ccdd Straight hair Dark eyes cd CcDd Wavy hair Dark eyes Ccdd Wavy hair Blue eyes ccdd Straight hair Dark eyes ccdd Straight hair Blue eyes Figure 7.2c
Extensions to Mendel s Laws. Genetics is not as simple as Gregor Mendel concluded, (one gene, one trait). We know now that there is a range of dominance and that genes can work together and interact. Ex: Incomplete dominance: When the F1 generation have an appearance in between the phenotypes of the parents. Ex: pink snapdragons offspring of red and white ones. Another way to say it is In incomplete dominance Heterozygote phenotype is somewhere between that of two homozygotes In humans hypercholesterolemia shows incomplete dominance.
Incomplete dominance in carnations
7.3 Extensions/modifications of Mendelian Genetics Incomplete dominance: two copies of the dominant allele are required to see the full phenotype; heterozygote phenotype is intermediate to the homozygotes (e.g., flower color in snapdragons) Flower color in snapdragons x = Red = RR White = rr Pink = Rr Figure 7.3
The Spectrum of Dominance Complete dominance occurs when phenotypes of the heterozygote and dominant homozygote are identical. Ex: having a widows peak or freckles. In incomplete dominance, the phenotype of F 1 hybrids is somewhere between the phenotypes of the two parental varieties In codominance, two dominant alleles affect the phenotype in separate, distinguishable ways. Ex: blood group type AB.
7.3 Extensions of Mendelian Genetics Codominance: neither allele is dominant to the other; heterozygote shows both traits at once (e.g., coat color in cattle) Coat color in cattle x = Red = R 1 R 1 White = R 2 R 2 Roan = R 1 R 2 (patchy red and white coat) Figure 7.4
Co-Dominance and multiple alleles: Co-dominance in human blood types Non-identical alleles specify two phenotypes that are both expressed in heterozygotes Having more than 2 alleles for a given trait and both alleles show in the phenotype. No single one is dominant over the other. Example: ABO blood types
7.3 Extensions of Mendelian Genetics Blood typing can be used to exclude potential parents. ABO blood group as three alleles of one gene: I A and I B are codominant to each other; i is recessive to both other alleles. An individual will have two of these alleles. Possible genotypes and phenotypes (blood types) are shown in the next slide.
The red blood cell antigen is coded for by the gene I (for isohaemaglutinogen) which has 3 alleles A,B and O
7.3 Extensions of Mendelian Genetics Blood typing could not be done on the decomposed skeletal remains. Red blood cell phenotype Type A Sugar A Red blood cell genotype I A I A or I A i Type B Sugar B I B I B or I B i Type AB Sugars A and B I A I B Type O ii Figure 7.5
Real life problem: A woman is suing her ex-husband for child support. She is blood group A (we don t know if she is homozygous or heterozygous) and her ex-husband is blood group B. The child is blood group O. Could he be the father of the child?
Mother could be either heterozygous: AO or homozygous AA Father could be BO or BB Answer: A AB BO O Probability of child being type O is one in four or 25% AO OO
Pleiotropy Most genes have multiple phenotypic effects, a property called pleiotropy For example, pleiotropic alleles are responsible for the multiple symptoms of certain hereditary diseases, such as cystic fibrosis, sickle-cell disease and hemophilia Pleiotropy: One genes having many effects. Only one gene affects an organism in many ways.
Pleiotropic effects of the sickle-cell allele in a homozygote
Great web sites to help you study http://www.biologymad.com/ http://www.kumc.edu/gec/ http://www.dnaftb.org/dnaftb/1/concept/
Abundance of a gene in a population is not related to dominance Other dominant traits: Polydactylism Astigmatism, near,far sightedness Hypertension Migrane headaches Hitchhikers thumb Hypercholesterolemia Tongue roller Free earlobes Dominant Traits Freckles Widow s peak Recessive Traits No freckles Straight hairline Free earlobe Attached earlobe Figure 9.8 A
Frequency of Dominant Alleles Dominant alleles are not necessarily more common in populations than recessive alleles For example, one baby out of 400 in the United States is born with extra fingers or toes The allele for this extra fingers/toes is dominant to the allele for the more common trait of five digits In this example, the recessive allele is far more prevalent than the dominant allele in the population
Dominant Recessive Coarse body hair Fine body hair Male pattern baldness Baldness Freckles Absence of freckles Astigmatism Normal vision Near or far sightedness Normal vision Normal hearing Deafness Broad lips thin lips Large eyes small eyes Polydactilism normal digits Feet with normal arches flat feet Hypertension normal blood pressure Diabetes normal excretion of insulin Migraine headaches normal Normal transport cystic fibrosis Hypercholesterolemia (familial) normal cholesterol levels
7.4 Sex Determination and Sex Linkage Prince Alexis suffered from hemophilia, the inability to clot blood normally due to the absence of a clotting factor. Gene for this clotting factor is on the X chromosome. Alexis inherited the hemophilia allele from his mother.
7.4 Sex Determination and Sex Linkage Humans have 22 pairs of autosomes and one pair of sex chromosomes Women: two X chromosomes Men: one X and one Y chromosome Male XY Meiosis Female XX X Y X X Possible sperm Possible eggs Fertilization This zygote will develop into a male. XY XX This zygote will develop into a female. Figure 7.6
Some disorders caused by recessive alleles on the X chromosome in humans: Color blindness Duchenne muscular dystrophy Hemophilia
SEX LINKED TRAITS ARE THOSE CARRIED BY THE X CHROMOSOME Red-Green color blindness Inability to see those colors. Red and green look all the same,like gray Hemophilia Blood clotting disorder. The clotting factor VIII is not made, individual can bleed to death. Duchenne Muscular dystrophy X linked recessive, gradual and progressive destruction of skeletal muscles. Faulty teeth enamel Extremely rare, X linked Dominant
7.4 Sex Determination and Sex Linkage Sex-linked genes: genes located on the sex chromosomes X-linked: located on the X chromosome Y-linked: located on the Y chromosome Males always inherit their X from their mother Males are more likely to express recessive X- linked traits than females Only females can be carriers of X-linked recessive traits.
7.4 Sex Determination and Sex Linkage Possible types of sperm Possible types of sperm (a) Unaffected male x Carrier female (b) Hemophiliac male x Unaffected female X H Y x X H X h X h Y x X H X H Possible types of eggs Possible types of eggs X H Y X H X H Unaffected female X H Y Unaffected male X H X h Carrier female X h Y Hemophiliac male 1 4 Unaffected females 1 4 1 Carrier females 4 Hemophiliac males 1 4 Unaffected males X h Y X H X h Carrier female X H Y Unaffected male 1 2 Carrier females 1 2 Unaffected males Figure 7.8
7.4 Sex Determination and Sex Linkage PLAY Animation X-Linked Recessive Traits Figure 7.10
What is the probability that the Russian Czar who was normal but married a hemophilia gene carrier have a daughter with hemophilia? A son with hemophilia? A grandchild with hemophilia from one of his daughters? Czar : Czarina:
7.4 Sex Determination and Sex Linkage Early female embryos randomly inactivate one of the X chromosomes in each cell. Inactivation is irreversible and inherited during cell division. It is caused by RNA wrapping around the X chromosome. Active X chromosome Muscular dystrophy gene Xist RNA gene Hemophilia gene 2 red-green color blindness genes Inactive X chromosome Xist RNA Figure 7.9
7.4 Sex Determination and Sex Linkage Result is patches of tissue in adult female with different X chromosomes active. (a) Phenotype Orange male Black female Tortoise shell female x = Genotype Allele for orange fur Allele for black fur (b) X inactivation Random X chromosome inactivation Early embryo Inactive X chromosome Active X chromosome Tortoiseshell cat with patches of orange and black Mitosis Mitosis Figure 7.10
Pedigree Analysis A pedigree is a family tree that describes the interrelationships of parents and children across generations Inheritance patterns of particular traits can be traced and described using pedigrees
7.5 Pedigrees Pedigree: a family tree, showing the inheritance of traits through several generations Symbols commonly used in pedigrees: Pedigree analysis symbols Female Male Marriage or mating Offspring in birth order (from left to right) or Affected individuals or Known or presumed carriers Figure 7.11
7.5 Pedigrees Pedigrees reveal modes of inheritance Pedigree for an autosomal dominant trait: (a) Dominant trait: Polydactyly pp Pp pp Pp pp pp Pp Pp Pp pp Two affected parents can have unaffected offspring. pp Two unaffected individuals cannot have affected offspring. Figure 7.12a
7.5 Pedigrees Pedigree for an autosomal recessive trait: (b) Recessive trait: Attached earlobes Ff ff FF ff Ff Ff Ff Ff Ff Ff Ff ff ff The recessive trait can skip a generation completely, producing unaffected individuals. ff ff ff ff Two affected individuals have affected offspring. Figure 7.12b
LE 14-14a Ww ww ww Ww First generation (grandparents) Ww ww ww Ww Ww ww Second generation (parents plus aunts and uncles) WW or Ww ww Third generation (two sisters) Widow s peak No widow s peak PowerPoint lecture prepared by Dominant trait (widow s peak) Steve McCommas Southern Illinois State University
7.5 Pedigrees Pedigree for an X-linked trait: (c) Sex-linked trait: Muscular dystrophy Affected offspring are most often male. X D Y X D X d Carrier X D X D X D Y X d Y X D X d X D Y X D Y X D Y X D X D X D Y Figure 7.12c
7.5 Pedigrees Pedigree analysis reveals that Queen Victoria s mother must have had a mutation for the hemophilia allele, which was ultimately passed on to Prince Alexis Romanov. Alice (Tsarina s mother) Queen Victoria Irene (Tsarina s sister) Princess Victoria Tsarina s grandmother first carrier of the mutant allele. Fred Tsarina (Tsarina s brother) Tsarina s great-grandmother underwent a mutation to the clotting factor VIII gene in her ovary during meiosis. Leopold (Tsarina s uncle) Waldemar Henry Son Alexis Tsarina s nephews Tsarina s daughters are all possible carriers. had hemophilia. Figure 7.13
DNA recombination or genetic engineering is the direct manipulation of genes for practical purposes DNA and Crime Scene Investigations DNA fingerprinting has provided a powerful tool for crime scene investigators DNA is isolated from biological fluids left at a crime scene The technique determines with near certainty whether two samples of DNA are from the same individual
7.6 DNA Fingerprinting No two individuals are genetically identical (except for MZ twins) Small differences in nucleotide sequences of their DNA This is the basis for DNA fingerprinting Unambiguous identification of people
7.6 DNA Fingerprinting Steps in DNA fingerprinting: DNA isolated from tissue sample DNA cut into fragments with enzymes DNAs of different sequences produce fragments of different sizes Fragments separated on basis of size and visualized Each person s set of fragments is unique
The tools of DNA fingerprinting Restriction enzymes (used to cut DNA fragments to do gel electrophoresis), PCR ( polymerase chain reaction)(used to make copies of DNA when there isn t enough) Taq polymerase enzyme (used to put together a copy of the DNA) Variable number tandem repeats (vntr) Gel electrophoresis- used to separate the fragments
7.6 DNA Fingerprinting Small amounts of DNA can be amplified using PCR (polymerase chain reaction) DNA is mixed with nucleotides, specific primers, Taq polymerase, and then is heated Heating splits the DNA molecules into two complementary strands Taq polymerase builds a new complementary strand DNA is heated again, splitting the DNA and starting a new cycle.
12.10 DNA Fingerprinting 1 st -The DNA molecule is cut with restriction enzymes 2 nd - we have to separate the fragments This is done by a technique called gel electrophoresis The DNA is placed on a tray filled with gel through which an electric current runs causing the fragments to move through the gel. The segments separate by how far they move in the gel according to size. The DNA will form bands corresponding to the bases (and no two people have the same sequence of bases) in the gel which are unique for each individual. This is DNA fingerprinting
7.6 DNA Fingerprinting PLAY Animation Polymerase Chain Reaction (PCR)
7.6 DNA Fingerprinting DNA is cut into fragments using restriction enzymes, which cut around DNA sequences called VNTRs (variable number tandem repeats) = Student 1 Variable number tandem repeat (VNTR) 4 VNTRs 5 VNTRs 6 VNTRs 3 VNTRs Student 2 Homologous chromosomes Figure 7.15
Restriction Enzymes Enzymes are used to "cut and paste" DNA DNA from two sources cut by restriction enzyme at specific restriction sites Resulting restriction fragments contain a doublestranded sequence of DNA with single-stranded "sticky ends" Fragments pair at their sticky ends by hydrogen bonding DNA ligase pastes the strand into a recombinant DNA molecule
7.6 DNA Fingerprinting Gel electrophoresis separates DNA fragments on basis of their sizes Electric current is applied to an agarose gel Smaller fragments run faster through the gel Fragments are transferred to a sheet of filter paper Labeled radioactive probe reveals locations of fragments containing VNTRs (variable number tandem repeats) These steps are illustrated in the next slide
7.6 DNA Fingerprinting Figure 7.16
12.10 Gel electrophoresis sorts DNA molecules by size Mixture of DNA molecules of different sizes Longer molecules Power source Gel Shorter molecules + + Completed gel Figure 12.10
7.6 DNA Fingerprinting Each person will have a unique pattern of bands. Figure 7.17
7.6 DNA Fingerprinting DNA fingerprinting showed that 9 persons were buried in the Ekaterinburg grave. Romanovs would be more similar in pattern to each other than to nonrelatives. All of a child s bands must be present in one or both of the parents.
7.6 DNA Fingerprinting Adult 1 Adult 2 Adult 3 Adult 4 Adult 5 Adult 6 Child 1 Child 2 Child 3 Figure 7.18
7.6 DNA Fingerprinting Pedigree of Romanov family DNA evidence Tsar s brother George Tsar Tsarina Carrier of hemophilia allele Tsarina s sister Not a carrier of hemophilia allele Olga Tatiana Maria Anastasia Alexis Hemophilia Members of Romanov family executed in 1918 Tsarina s niece Alice DNA evidence Tsarina s grandnephew Prince Philip Queen Elizabeth II Lady Diana Charles Anne Timothy Lawrence Andrew Sarah Ferguson Edward Sophie Rhys-Jones William Henry Peter Zara Beatrice Eugenie Louise Figure 7.20
7.6 DNA Fingerprinting To see if parents and their children were Romanovs, DNA fingerprints were prepared for relatives of tsar and tsarina. Adult male skeleton (related to the children) was related to George, the tsar s brother. Adult female skeleton (related to the children) was related to Prince Philip, the tsarina s grand-nephew. Conclusion: the grave contained the tsar, tsarina, three of their children, and four servants.
DNA and Crime Scene Investigations Many violent crimes go unsolved For lack of enough evidence If biological fluids are left at a crime scene DNA can be isolated from them
Defendant s blood (D) Blood from defendant s clothes Victim s blood (V)
How Restriction Fragments Reflect DNA Sequence Restriction fragment length polymorphisms (RFLPs) Reflect differences in the sequences of DNA samples Crime scene Suspect w Cut C C G G G G C C z A C G G T G C C x y Cut C C G G G G C C y Cut C C G G G G C C Figure 12.11A DNA from chromosomes
7.6 DNA Fingerprinting Each cycle, the amount of DNA doubles. Primer 1 PCR is used to amplify, or make copies of, DNA. During a PCR reaction, primers (free nucleotides) and DNA are mixed with heat-tolerant polymerase. Double stranded DNA Primer Polymerase 4 A copy of the DNA template is assembled. 2 The DNA is heated to separate, or denature, the two strands. 5 The mixture is heated again. The process is repeated many times, doubling the DNA amount each time. 3 As the mixture cools, the primers bond to the DNA template and the two polymerase use the primers to initiate synthesis. Figure 7.14
Detecting a harmful allele using restriction fragment analysis 1 Restriction fragment preparation I II III 2 Gel electrophoresis Restriction fragments I II III 3 Blotting Filter paper 4 Radioactive probe Radioactive, singlestranded DNA (probe) Probe 5 Detection of radioactivity (autoradiography) I II III Film Figure 12.11C I II III