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Biology 2581B Lecture Notes - Lecture 12: Single-Nucleotide Polymorphism, Southern Blot, Craig Venter

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Western University
Department
Biology
Course
Biology 2581B
Professor
David R Smith

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Detection of Mutants and Genetic Variants
Readings:
- Chapter 15 p 489-505
Key Concepts:
- Single Nucleotide Polymorphisms
- Short repeated sequences
- Minisatellites and fingerprinting
How do individuals differ within a species?
- Not all individuals within a species have identical genomes. There are slight variations.
How distinguish between genetic variation, a wild type allele and a mutation?
- Rather than calling it a wild type with mutations, we call it variations.
- They are more or less equal and representative in population. It is hard to say which one
is the wild type.
- Polymorphic → a locus with two or more alleles where each allele is present in more
than 1% of the populations
- There is more than one variation which seems to be representative of that particular
gene just in different type of individual
- Alleles of a polymorphic locus are called genetic variations rather than wild type or
mutants.
- Not equally represented (frequency might not differ)
- There do not seem to be an immediate impact b/c these variations are present in
different individuals that look healthy/fine.
- Advanced sequence technologies allows sequencing of complete genomes from
individuals
o Craig Venter
o James Watson
- 2 021 206 single nucleotide substitutions
- 5 015 single nucleotide substitutions cause amino acid changes of expressed proteins
- There is a lot of different variation between individuals and there can be a huge number
of SNPs within a population which can cause AA changes if they are coding sequences.
- Compare how many of these
changes are unique and how many
of these changes are shared by
different individuals.
- There isn’t a big change between
these 2 guys to someone who has a
different ethnic background.
- Amino acid changing substitutions:
Similar distribution but numbers are
smaller because base substitutions
do not necessarily lead to an amino
acid substitution.
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- Single nucleotide substitutions in the genomic region for the cystic fibrosis gene
- Most of them do not have any effect on function
- Only approx. 2% of the nucleotides in this region are part of coding sequences
- Not only base substitutions
- Over 100,000 deletion and insertions ranging from 2 to 37,896 bp
- Overall genome length differ as much as 1% between healthy individuals
- .≠ Every insertion/deletion that can be tolerated … depends on where they are occurring.
Loci
- Any location within the genome that has a defined chromosomal location
- irrespective of function
- coding or non-coding
- short or long
- single base pair substitution – one gene – several genes
- Allele → genetic variation at a specific locus within the particular gene
Polymorphic loci:
- Single Nucleotide Polymorphism: SNPs or snips
- Insertions and Deletions: InDels or DIPs
- Simple Sequence Repeats: SSRs
- Copy Number Polymorphisms: CNPs duplicate a certain sequence, might be involved in
the gene
o Could have a 3rd copy somewhere in the genome
- Complex variants = none of the above
SNPs
- SNPs are the most common genetic
variation
- Spontaneous – during replication OR
- Induced – chemical mutagen
- SNPs are base pair substitutions
- For a diploid genome:
o Homozygous T: TT
o Heterozygous T: TC
o Homozygous C: CC
How to detect SNPs
1. Southern Blot analysis of restriction site altering SNPs
- Combine restriction enzyme cutting + Use probe to a specific region
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SNP allele 1: There are 3 recognition site for EcoRI. When EcoRI cuts at these recognitions
sites, you get different fragments
- Interested in blue highlighted AT/GC base pair.
- Probe recognizes sequence on restriction fragment.
o Only highlight that fragment on a southern blot, nothing else.
o Everything else do not have complementary sequences to the probe.
o Note: Probe length ≠ Length of the restriction
fragment. Probe just has to be efficiently long
to recognize and base pair with sequence on
this particular fragment.
SNP allele 2: Base pair substitution from AT → GC.
- Changes recognition site of EcoRI. Enzyme
cannot cut here anymore.
o Enzymes are very specific & recognize
specific sequences.
- When EcoRI cuts, b/c you lack a restriction cut site, the fragment will be longer.
- The probe still recognizes this fragment.
- In the southern blot, there is a size difference between the two restriction fragments
depending on what allele is present.
- Homozygous for SNP allele 1: Just the small
fragment.
o Homozygote who only has SNP allele 1 in
both of his chromosomes.
- Homozygous for SNP allele 2: Just the larger
fragment. The fragment that is generated when
you cut DNA from a person like that is larger b/c
restriction cut site lost.
o Homozygote who only has SNP allele 2 in
both of his chromosomes.
- Heterozygote: Visualize 2 different fragments
generated by 2 chromosomes
o Heterozygous with SNP allele 1 on one chromosome and SNP allele 2 on the other
chromosome.
- Method allows you to detect if a particular person has a single base pair change in a
particular locus in a particular sequence but you have to know a lot of things about it.
2. PCR analysis of restriction site altering SNPs
- SNP allele with restriction site sequence for MstII RE.
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Document Summary

Not all individuals within a species have identical genomes. Rather than calling it a wild type with mutations, we call it variations. They are more or less equal and representative in population. It is hard to say which one is the wild type. Polymorphic a locus with two or more alleles where each allele is present in more than 1% of the populations. There is more than one variation which seems to be representative of that particular gene just in different type of individual. Alleles of a polymorphic locus are called genetic variations rather than wild type or mutants. Not equally represented (frequency might not differ) There do not seem to be an immediate impact b/c these variations are present in different individuals that look healthy/fine. Advanced sequence technologies allows sequencing of complete genomes from individuals: craig venter, james watson. 5 015 single nucleotide substitutions cause amino acid changes of expressed proteins.

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Related Questions

1. Humans and chimpanzees share several polymorphic loci. Which is the LEAST plausible explanation for this fact?

A. Balancing selection has maintained polymorphism in both species

B. Incomplete lineage sorting left polymorphic loci and those have persisted since the speciation event.

C. Interbreeding has allowed introgression of alleles at theses loci in both directions.

D. Genetic variation provided by the polymorphic alleles enhances resistance to pathogens.

2. According to the social brain hypothesis, we should expect primates living in larger social groups to have:

A. Larger brains, specifically, a neocortex of relatively larger size

B. Lager incisors and canines, to defend against more social competitors

C. Smaller brains, because many individuals can work together to solve problems

D. Great variation in brain and neocortex size, with the dominant individuals having the largest

3. You extract DNA from human bones in an ancient village buried by volcanic ash. You discover that there is an extremely high frequency of the LCT allele for lactase persistence. What might you reasonably conclude about the population?

A. This population is likely the source of the high frequency of the LCT gene in Northern Europe

B. The village could not be more than 4,500 years old

C. The members of this population likely used domesticated livestock for their milk

D. villagers had a diet rich in wheat or another domesticated crop

4. Why do populations father from Africa have more deleterious mutations in their genome?

A. Repeated bottlenecks and founder effects made drift more intense more often

B. Populations farther from Africa have been exposed to more mutagens in the environment

C. As people migrated over the globe they interbred with Neanderthals and Denisovans

D. The populations are younger, so purifying selection has not had time to purge delirious mutations from the populations.

1.. Polymorphisms for a given trait are relatively common in most species. Discuss the possible advantages or polymorphisms in terms of evolutionary fitness. Be sure to relate your discussion to a specific example. It is also possible that organisms showing more heterozygosity may actually be less fit. Describe one such possible scenario.

2. In the peppered moth (Biston betularia), black individuals may be either homozygous (A1A1) or heterozygous(A1A2), whereas the pale gray moths are homozygous (A2A2). Suppose that in a sample of 250 moths from one locality, 108 are black, and 142 are gray.

3. Retrace the history of primate evolution from early primates to monkeys, apes and hominins. When did some ofthe significant divergences occur (such as between the prosimians and the monkeys or between the chimps and hominins)? What was happening with the continents and the major climatic conditions during these times?What are the major species (including the extinct species) along the human lineage after the split with the chimps and bonobos? Where did many of these events occur on the globe and when?

4. Spencer Wells described his study how the genetic markers in humans are being used to infer the historical geographical distribution of humans and the dispersal of our ancestors. Explain how the genetic markers on our DNA (from blood samples) helps us understand that history. What are the techniques he is using? What is the nature of the evidence? What are the major conclusions about the journey of our ancestors out of Africa?What were the major migrations of humans to all the other continents? Approximately when did each migration occur and what were the major paths? How does the evidence support these conclusions? What Supporting evidence besides the DNA evidence is being used to support these conclusions?

5. Provide an adaptive and a non adaptive hypothesis for the evolutionary loss of useless organs, such as eyes in many cave-dwelling animals. How might these hypotheses be tested?

6. Directional selection is one of the models of natural selection. Describe this type of selection in terms of the frequencies of advantageous alleles over time. What is the trend over time? What is the eventual outcome for this allele as long as no other evolutionary factors intervene? Describe at least one good example of directional selection.

7. Consider the first copy of an allele for insecticide resistance that arises by mutation in a population of insects exposed to an insecticide. Is this mutation an adaptation? If, after some generations, we find that most of the population is resistant, is the resistance an adaptation? If we discover genetic variation for insecticide resistance in a population that has had no experience of insecticides, is that variation an adaptation? If an insect population is polymorphic for two alleles, each of which confers resistance against one of two pesticides that are alternately applied, is that variation an adaptation? Or is each of the two resistance traits an adaptation?Explain your reasoning thoroughly.

8. Suppose that a mutation in a species of annual plant increases allocation to chemical defenses against herbivores, but decreases production of flowers and seeds (i.e., there is an allocation trade-off). What would you have to measure in a field study in order to predict whether or not the frequency of the mutation will increase?

9. In many socially monogamous species of parrots and other birds, both sexes are brilliantly colored or highly ornamented. Is sexual selection likely to be responsible for the coloration and ornaments of both sexes? How can there be sexual selection in pair-bonding species with a 1:1 sex ratio, since every individual presumably obtains a mate? Which of the models of sexual selection described in the book might account for these species characteristics?

10. Kin selection explains why organisms may provide benefits to relatives. Is there a conflict between the principle of kin selection and the evolution of siblicide and abortion?

11.Many species of albatrosses and other seabirds nest in colonies on islands. The adult search for food at sea,sometimes over great distances. Some ecologists who study seabirds turn to their favorite topic of discussion in a bar one evening, and one says, “I can imagine an albatross genotype that destroys the eggs of nestlings of its colony mates when the parents are away foraging, and leaves them dead without eating them. Do you think such a behavior would evolve?” One of his companions says, “Yes, because it would make more food available for the albatross and its offspring.” Another says, “No, because it would threaten the survival of the population.” The fourth says, “You’re both wrong. I have a different explanation for why albatrosses don’t kill others’ chicks.” What is the fourth ecologist’s explanation, and why does she think her companions are wrong?

12. The concept of a species is fundamental to understanding biology, yet there may not be one definition that fits all “types” of organisms (e.g. animals, plants, fungi, bacteria, slime mold, lichens, etc.). Discuss several of the definitions of a species as presented in class and present your own definition for a species. Compare the strengths and weaknesses of each definition. Ernst Mayr is strong proponent of the biological species concept.How does he justify his position? Do you agree or disagree? If we employ the biological species concept, when did species first exist? What were organisms before then, if not species? What might the consequences of the emergence of species be for processes of adaptation and diversification? Be sure to explain your reasoning thoroughly.

13. The process of speciation ultimately involves the development of reproductive isolation between two related populations of organisms. Describe the various types of both prezygotic and postzygotic reproductive isolation and give an example of each.

14. Respond to the following quote from the character named Mr. Spock in “Star Trek”:“A truly successful parasite is commensal, living in amity with its host, or even giving it positive advantages, as for instance, the protozoans who live in the digestive system of your termites and digest for them the wood they eat. A parasite that regularly and inevitably kills its host cannot survive long, in the evolutionary sense, unless it multiplies with tremendous rapidity ... it is not pro-survival.”From what you have learned in this course, do you agree or disagree with Spock? How can you apply your reasoning to the relative “fitness” of HIV which causes AIDS, the Ebola virus which causes hemorrhagic fever,fleas which suck blood from dogs, or the prokaryotic ancestors of mitochondria and chloroplasts?

15. In many species of birds and mammals, clutch size is larger in populations at high latitudes than in population sat low latitudes. Species of lizards and snakes at high latitudes often have smaller clutches, however, and are more frequently viviparous (bear live young rather than laying eggs), than low-latitude species. What selective factors might be responsible for these patterns? Explain your reasoning.

16. The popular media often presents evolution as being a predictable process with a definite goal. For instance, in one “Star Trek: Voyager” episode, the captain instructs the ship’s computer to extrapolate the “probable course” of evolution of hadrosaurs (a bipedal dinosaur), if hadrosaurs had been removed from Earth before the Cretaceous-Tertiary (K-T) Extinction and allowed to evolve on another planet. What information about the hadrosaurs’ new environment would have been useful in developing the best possible prediction? Given what you know on genetic drift and selective agents, is it possible to accurately predict the long-term course of evolution?

17. Young children often develop strong food preferences that sometimes seem bizarre to their parents --- such as wanting to eat only a certain food prepared in a familiar way, or being reluctant to each new foods or foods with strong or bitter tastes (like coffee and some vegetables). Develop an evolutionary explanation for such preferences. What evidence would you gather to investigate your hypothesis and rule out alternative explanations?

18. Many organisms, such as mammals, only reproduce by sexual means. Other organisms are exclusively asexual,such as many of the bacteria and fungi. And still others have ways of propagating by both sexual and asexualmeans. Discuss the relative advantages and disadvantages of each mode of reproduction. Why aren’t all organisms sexual critters? Why are many asexual organisms still very successful as evidenced by the pure abundance and diversity? What is the adaptive significance of having different genders (e.g. male and female)?In other words, why sex?

19. How might differential expression and regulation by Hox genes contribute to mosaic evolution in which different segments of an animal body plan evolve different morphology?

20. Development of a morphological structure involves many different types of gene products, including transcription factors, signaling proteins, and “effector” genes such as enzymes. When a morphological change occurs in a single mutational step, which of these types might be more or less likely to be involved? Within a gene, would such single-step events be more likely to involve coding or non-coding sequences, and what characteristics of the gene’s function might affect this likelihood?

21. Since 2001, the complete DNA sequences of the genomes of many people have been determined. If humans,along with other forms of life, have evolved from a common ancestor, what evidence of this would be expected in the human genome? In what ways might the history and processes of evolution help us interpret and make sense of the human genome sequence data?

22. Paleontologists and some biologists commonly infer function, and even behavior, from anatomical details.Skeletal features, for example, are often used to infer that an extinct mammal (such as an early hominid) was highly, somewhat, or not at all arboreal. This inference assumes a good fit of form to function (i.e., optimal form). Can this assumption be justified?

23. Stephen Jay Gould (1989) and others have argued that the evolution of self-conscious, intelligent species (i.e.humans) was historically contingent: it would not have occurred had any of a great many historical events been different. The philosopher Daniel Dennett (1995) and others have disagreed, arguing that convergent evolution is so common that if humans had not evolved, some other lineage would probably have given rise to a species with similar mental abilities. What do you think, and why? If Gould’s position is right, what are its philosophical implications, if any?

24. Explain how Intelligent Design was determined as a religious view rather than a scientific view in the court casein Dover, PA. Should a scientific explanation of evolution be included in a science curriculum in K-12 schools?How is science and religion different in how they seek answers to questions? How is term "theory" used differently in science than in everyday English use? What is biological evolution? What evidence is used to support the scientific explanation of evolution? Is it possible to accept scientific explanations without rejecting religious faith?

25. Discuss how a creationist versus an evolutionary biologist might explain some human characteristics and the implications of their differences. Sample characteristics: eyes; wisdom teeth; individually unique friction ridges(fingerprints); five digits rather than some other number; susceptibility to infections; fever when infected;variation in sexual orientation; limited life span.

Expression vectors inprokaryotes do not make functional eukaryotic gene products inbacteria very well because

Answer Not sure which one ?!

· the codon sequence for prokaryotes is differentthan the codon sequence in eukaryotes

· there are no disulfide bridges formed in proteinsnormally made in prokaryotes

· prokaryotic expression vectors cannot translateeukarytic sequences

· eukaryotic genes have introns, and prokaryotesdon't

· eukaryotic genes have exons and prokaryotesdon't


I create a knockout mouseusing the agouti/black fur system \. When I cross the agoutioffspring of the originally engineered mouse, I find a ratio of 2agouti mice to 1 black furred mouse. What is the bestexplanation?

Answer not sure which one?!

· The gene knocked out was recessive.

· The gene knocked out was dominant.

· The gene knocked out was a lethalgene.

· The knockput was integrated into a random spot,and did not knockput the original gene.

A restriction enzyme cuts DNAand leaves the following end

xxCTGCA
xxG

Which of the following could be the sequence of the correspondingend of the other end of the cut DNA?

Answer

· xxG
xxCTGCA

· xxC
xxGTGCA

· xxCCGAT
xxG

· xxGGCTA
xxC


An SNP always occurs dueto

Answer

· a one base-pair change.

· a three base-pair change.

· a single restriction site change.

· a single amino acid change.

· None of these.


I PCR out a mutated gene in apatient with Lisenbee chorea (the inability to dance in acoordinated fashion) and I compare it to another patient with thatsame disease phenotype. One subject had a mutation on chromosome 4,and the other subject couldn't rock it because of a mutation onchromosome 5. This is an exampe of

Answer

· pleitropy

· locus heterogeneity

· allelic heterogeneity

· dominant negative mutation

· penetrance

In his experiments, Mendelnoted that when two traits are involved in a genetic cross, theyare inherited independently of each other. Though Mendel didn'tknow about chromosomes, this still holds true (mostly)because

Answer

· genes on the same chromosome are often mixedthrough meiotic crossover during the formation ofgametes.

· variant alleles of the same gene do not interferewith the phenotypic expression of the gentoype in different geneticloci.

· most higher organisms are diploid, making only twoalleles available for every genetic locus, and there is a 50/50chance of geting either allele from a single parent.

· expression of phenotypes can vary from thegenotypes because of encironmantal influences.

· chromosomes often recombine

Anticipation is caused by amutation that increases in expressivity over subsequentgenerations.

Answer

· True

· False

Question 44

I have a genotype that shouldproduce a specific phenotype, but some of the individuals with thegenotype do not demonstrate any evidence of the phenotype. I wouldconsider this an example of

Answer

· allelic heterogeneity.

· locus heterogeneity.

· penetrance.

· expressitivity.

· sheer luck.

Question

Which of the following doesnot occur during the PCR reactions?

Answer

· Changes in the reaction temperature by athermocycler

· Denaturation at high temperatures

· Synthesis of oligonucleotide primers

· Extension of primers by DNA polymerase

· Annealing of oligonucleotide primers

Question

DNA markers, or variantnon-coding regions of DNA, are DNA polymorphisms that are usefulfor genetic mapping.

Answer

· True

· False

A mosaic is an organismwith

Answer

· multiple genotypes within one organism

· multiple alleles within one genotype

· more than one color of fur

· transgenes added to the zygote beforedevelopement

· a wt phenotype but a mutated genotype

Question

Genotype causesphenotype.

Answer

· No, gentoype just influences phenotype.

· Yes, genotype is the DNA sequence that createsphenotype.

Question

A true genetic chimera can becreated by

Answer

· mutating a gene early on in the development of anorganism resulting in different alleles being present in theadult

· multiple fertilized eggs or zygotes fusing to formone embryo

· adding a transgene to the genome of an organismduring fetal development only

· adding cells of a different species to an adultorganism

· adding a transgene to the genome of an animal atany stage in development

Question

The ABO blood group can bestbe explained by the concept of _______.

Answer

· dominant traits

· recessive traits

· allelic heterogeneity

· locus heterogeneity

· vampirism

If a loss of functionmutation creates a dominant phenotype, it may be becauseof

Answer

· haploinsufficiency

· penetrance

· expressivity

· allelic heterogeneity

· locus heterogeneity

Please select the best matchfor each.

Answer

· PCR: Technique used to generatecopies of a DNA fragment

· Southern blot : Screeningprocedure used to assay DNA fragments

· RFLPs : DNA fragments generatedby restriction enzymes that vary in length betweenindividuals

· Northern blot : Screeningprocedure used to assay mRNA fragments

· Electrophoresis Movement ofmacromolecules in an electrical field

Answer

A.

Screening procedure used toassay DNA fragments

B.

Movement ofmacromolecules in an electrical field

C.

Screening procedure used toassay mRNA fragments

D.

DNA fragments generated byrestriction enzymes that vary in length betweenindividuals

E.

Technique used to generatecopies of a DNA fragment

A gene mutates, and theprotein produced has a novel way of interacting with the cell, andcreates a new phenotype because of this new functionality. This iscalled

Answer

· pleitropy

· locus heterogeneity

· allelic heterogeneity

· dominant negative mutation

· gain of function dominant mutation

Question 62

Mutations in the somaticchromosomes may be inherited by the next generation.

Answer

· True

· False


A degenerate PCR primer withmany variant sequences must be used to make multiple copies of agene

Answer

· if only the protein sequence of the gene productis known to construct the primers

· if the DNA you are using is cDNA to constructthe primers

· if the DNA you are using is genomic DNA to becopied

· if the DNA you are trying to copy iscDNA

· if the vector is prokaryotic and the transformedcell is eukarytotic

Question

Please select the best matchfor each term.

Answer

· Restriction enzyme


· PCR C

· Plasmid

· YAC A

· Dideoxynucleotide

Answer

A.

Endonuclease isolated frombacteria

B.

Eukaryotic cloningvector

C.

Method for amplification ofDNA sequences

D.

Small circular fragment ofDNA

E.

Molecule used in DNAsequencing

Question

A couple goes on MauryPovitch, and the results are in: you are not the father. But noother man impregnated the female (granted, unlikely for a MauryPovitch guest, but work with me here) and he must be the father.DNA analysis claims otherwise, though the child definitely wasmom's (poor thing). What may be going on here?

Answer

· the child is parthenogenic because the motheractually impregnated herself like a shark, and the child's DNA isall mom's

· the child had a mutation that changed the genethat is used to trace paternity

· the child is a mosaic because he is actually a setof twins fused early during fetal development, and therefore camefrom 2 eggs and 2 sperm

· the dad may have germ-line mosaicism, meaning thatthe genotype of his sperm is different from his somaticgenotype

· mitochondrial DNA only comes from mom, so there isno way to tell whobthe baby's father is