CHAPTER 22 QUESTIONS

AFLP:

- Amplified restriction fragment length polymorphism - Same as an RFLLP except that the site is amplified via PCR instead of isolating the chromosomal DNA

S2: An RFLP marker I located 1 million bp away from a gene of interest. Your goal is to start at this RFLP marker and walk to this gene. The average insert size in the library is 55,000 bp, and the average overlap at each end is 5,000 bp. Approximately how many step will is take to get there?

-Each step is only 50,000 bp (55,000-5,000= 50,000), because you have to subtract the overlap between adjacent fragments, which is 5000 bp, from the average inset size. Therefore, it will take about 20 steps to go 1 million bp-

1. In what year did the Human Genome Project officially begin?

1990

A. What is the Arrangement of these two STSs in this individual? B. What is the map distance between STS-1 and STS-2?

A. 423, 234 and 322, 198 are the most common so... STS-1: 234, 198 and STS-2: 423, 322 B. 4 recombinant sperm in lanes 15, 22, 25, 38 Map distance: recombinant/total = 4/40 * 100 = 10.0 mu

C2: For each of the following, decide is it could be appropriately described as a genome: A. The E. coli chromosome B. Human chromosome 11 C. A complete set of 10 chromosomes in corn D. A copy of the single-stranded RNA packaged into human immunodeficiency virus (HIV)

A. Yes B. No C. Yes D. Yes

E1: Would the following methods be described as linkage, cytogenetic, or physical mapping? A. Fluorescence in situ hybridization (FISH) B. Conducting dihybrid crosses to compute map distances. C. Chromosome walking. D. Examination of polytene chromosomes in Drosophila E. Use of RFLPs in crosses F. Using BACs to construct a contig

A. cytogenetic mapping B. linkage mapping C. physical mapping D. cytogenetic mapping E. linkage mapping F. physical mapping

1. Shotgun sequencing is a method of DNA sequencing in which... A. the DNA fragments to be sequenced are randomly generated from larger DNA fragments B. the sequencing reactions are carried out in rapid succession C. the samples to be sequenced are rapidly generated by PCR D. all of these occur

A. the DNA fragments to be sequenced are randomly generated from larger DNA fragments

3. A prokaryotic genome is about 4 million bp in length. About how many genes would you expect it to contain? A. 400 B. 4000 C. 40000 D. 400000

B. 4000

1. What is a contig? A. a fragment of DNA that have been inserted into a vector B. a series of vectors that contain inserts that have overlapping regions of chromosomal DNA C. A method of identifying a disease-causing allele D. a method of sequencing DNA

B. a series of vectors that contain inserts that have overlapping regions of chromosomal DNA

5. Based on the information in the table, why is it difficult to accurately determine the genome size of eukaryotic genomes? .

Because the DNA in certain heterochromatic regions cannot be sequences so the total amount is difficult to estimate

1. A molecular marker is a ____ found at a specific site on a chromosome that has properties that allow it to be ____. A. colored dye, visualized via microscopy B. colored dye, visualized on a gel C. segment of DNA, uniquely identified using molecular tools D. segment of DNA, visualized via microscopy

C. segment of DNA, uniquely identified using molecular tools

2. Which of the following is an example of a molecular marker? A. RFLP B. Microsatellite C. Single-nucleotide polymorphism D. All of the above

D. All of the above

3. To map the distance between molecular markers via crosses, the markers must be A. polymorphic B. monomorphic C. fluorescently labeled D. on different chromosomes

D. on different chromosomes

4. Look at table 22.3. Is there a proportional change in the number of genes as the size of the eukaryotic genomes increase?

No, it generally stays the same. Approximately 1000 bp in 1000000.

1. What type of mapping replies on microscopy? a. cytogenetic mapping b. linkage mapping c. physical mapping d. all of the above

d. all of the above

What are the steps to FISH?

o Cells are treated with agents that make them swell and fixes them onto slides o Probe hybridizes to the denatured chromosomal DNA only at specific complementary sites in the genome -Note that the chromosomes are highly condensed metaphase chromosomes that have already replicated >sister chromatids -Each x-shaped chromosome actually contains two copies of a particular gene -Because the sister chromatids are identical, a probe that recognizes one sister chromatid will also bind to the other o To the denatured DNA, a single-stranded DNA probes are added that have biotin incorporated into them o Fluorescently labeled avidin that binds to biotin is added -Fluorescent molecule is added to the probe o View with a fluorescent microscope

STS:

- Sequence-tagged site - General term to describe any molecular marker that is found at a unique site in the genome and is amplified by PCR - AFLPs. Microsatellites, and SNPs can provide sequence-tagged sites within a genome

E10: Describe the molecular features of a BAC cloning vector. What is the primary advantage of a BAC over a plasmid or viral vector?

-BACs are bacterial artificial chromosomes that are developed from bacterial F factors. They can contain up to 300,000 bp and sometimes larger. They are also somewhat easier to use than YACs because the DNA is inserted into a circular molecule and transformed into E. coli. Their molecular features are that they are vectors that contain several genes that function in vector replication and segregation. They have an rorigin of replication (oriS) which are encoded by a gene called the repE gene. They contain parA, parB, parC genes that encode proteins required for the proper segregation of the vector into daughter cells, chloramphenicol resistance gene, cmR, provides a way to select for cells that have taken up the vector based on their ability to grow in the presence of the antibiotic chloramphenicol. They contain unique restriction enzyme sites, such as HindIII, BamHI, SphI, for the insertion of large fragments of DNA that are located within the lacZ gene, which encodes the enzyme β-galactosidase. Vectors with DNA inserts can be determined by plating cells on media containing the compound X-Gal-

2. What does is mean if a molecular marker is polymorphic? Why is this an important characteristic?

A polymorphic marker is a type of molecular marker used with alleles within a population that may vary from individual to individual. The distance between linked molecular markers can be determined from the outcomes of crosses. Molecular techniques allows researchers to find it easier to identify many molecular markers within a given species genome rather than identifying many allelic differences among individuals and because of this, geneticists have increasingly turned to molecular markers as points of reference along genetic maps. Detailed genetic maps can be constructed in which a series of many molecular markers have been identified along each chromosome of certain species like humans, model organisms, agricultural species, and many others.

2.Which of the following was not a goal of the Human Genome Project? A. To obtain the DNA sequence of the entire human genome B. To successfully clone a mammal C. To develop technology for the management of human genome information D. To analyze the genomes of model organisms

B. To successfully clone a mammal

E16: When conducting physical mapping studies, place the following methods in their most logical order: A. Clone large fragments of DNA to make a BAC library. B. Determine the DNA sequence of subclones from a cosmid. C. Subclone BAC fragments to make a cosmid library D. Subclone cosmid fragments for DNA sequencing

-A, C, D, B-

E5: Figure 22. 2 describes the technique of FISH. Why is it necessary to "fix" the cells (and the chromosomes inside of them) to the slides? What does it mean to fix them? Why is it necessary to denature the chromosomal DNA?

-Fixing means that the cell is chemically frozen. The contents of the cell cannot mix together with other cells and its morphology does not change. To fix means that the cell is adhered to the slide and it cannot move. It is necessary to denature the chromosomal DNA for the probe to bind to it. Since the probe is complementary to the DNA strand, it can only bind to it if it is denatured-

S3: Does a molecular marker does not have to be polymorphic to be useful in mapping studies? Does a molecular marker have to be polymorphic to be useful in linkage mapping (i.e. involving family pedigree studies or genetic crosses)? Explain why or why not.

-A molecular marker does not have to be polymorphic to be useful in physical or cytological mapping studies. Many STSs that are used in physical or cytogenetic studies are monomorphic. Monomorphic markers can provide landmarks in such mapping studies. In linkage mapping studies, however, a marker must be polymorphic to be useful because they can be RFLPs, microsatellites, or SNPs. To compute map distances in linkage analysis, individuals must be heterozygous for two or more markers (or genes). For experimental organisms, heterozygotes are testcrossed to homozygotes, and then the number of recombinant offspring and nonrecombinant offspring are determined. For markers that do not assort independently (i.e. linked markers), the map distance is computed as the number of recombinant offspring divided by the total number of offspring times 100-

1. The technique of fluorescence in situ hybridization involves the use of a _____ that hybridizes to a _______. A. radiolabeled probe, band on a gel B. radiolabeled probe, specific site on an intact chromosome C. fluorescent probe, band on a gel D. fluorescent probe, specific site on an intact chromosome

D. fluorescent probe, specific site on an intact chromosome

2. What was the source of the DNA used in this project?

DNA used in this project was obtained from human volunteers, their identity was not revealed to protect their privacy.

3. What causes and RFLP to be polymorphic? What causes STRs to be polymorphic?

RFLPs are polymorphic because the restriction site distances varies among individuals. In STRs, the length of the short sequences that are repeated in a row varies in a range, but it is not a fixed value.

SNP:

- Single-nucleotide polymorphism - Site in the genome where a single nucleotide is polymorphic among different individuals - These sites occur commonly in all genomes, and they are gaining greater use in the mapping of disease-causing alleles and in the mapping of genes that contribute to quantitative traits that are valuable in agriculture

STR/microsatellites:

- Site in the genome that contains many short sequences that are repeated many times in a row - The total length is usually in the size range of 50-200 bp, and their lengths may be polymorphic within a population - They are isolated via PCR - Microsatellites are also called short tandem repeats (STRs) and simple sequence repeats (SSRs)

E8: What is a contig? Explain how you would determine that two clones in a contig are overlapping?

-A contig is a series of clones that contains contiguous, overlapping pieces of chromosomal DNA. In order to determine that two clones in a contig are overlapping one would either use Southern blotting methods or molecular markers. The use of computer programs allows researchers to identify regions that are potentially overlapping-

3. Explain the basic approach of shotgun sequencing.

Shotgun sequencing is found to be the most efficient and inexpensive way to sequence genomes. DNA fragments to be sequenced are randomly generated from larger DNA fragments and genomic DNA is isolated and broken into smaller DNA fragments, typically 1500 bp or longer in length. Researchers then randomly sequence such fragments from the genome and by chance, some fragments overlap. This allows researchers to order them as they are found in the intact chromosome. An advantage of shotgun sequencing is that it does not require extensive mapping which can be very time-consuming and expensive. A disadvantage is that researchers waste time sequencing the same region of DNA more times than needed. To calculate the probability that a base will not be sequenced one muse use this equation: P=e^-m.

C3: Which of the following statements are true about molecular markers? A. All molecular markers are segments of DNA that carry specific genes. B. A molecular marker is a segment of DNA that is found at a specific location in a genome. C. We can follow the transmission of a molecular marker by analyzing the phenotype (i.e. the individual's bodily characteristics) of offspring. D. We can follow the transmission of molecular markers using molecular techniques such as gel electrophoresis. TRUE E. An STS is a molecular marker.

A. FALSE, they do not carry genes B. TRUE C. FALSE, the marker cannot a gene that affects phenotype. D. TRUE

E14: Take a look at solved problem S1. Let's suppose a male is heterozygous for two polymorphic sequence-tagged sites. STS-1 exists in two sizes: 211 bp and 289 bp. STS-2 also exists in two sizes: 115 bp and 422 bp. A sample of sperm was collected form this man, and individual sperm were placed into 30 separate tubes. Into each of the 30 tubes were added the primers that amplify STS-1 and STS-2, and then the samples were subjected to PCR. The following results were obtained... A. What is the arrangement of these STSs in this individual? B. What is the linkage distance between STS-1 and STS-2? C. Could this approach of analyzing a population of sperm be applied to RFLPs?

A. 422, 289: 15 422, 211: 1 211, 115: 13 211 289: 1 STS-1: 289, 211 and STS-2: 422, 115 B. 2 recombinants, 30 total 2/30 *100= 6.67 mu C. Yes, but there is not enough DNA in one sperm to carry out an RFLP analysis unless the DNA is amplified by PCR

E19: A human gene, which we will call gene X, is located on chromosome and is found as a normal allele and a recessive disease-causing allele. The location of gene X gas been approximated on the map shows here that contains found STSs, labeled STS-1, STS-2, STS-3, and STS-4. STS-1 STS-2 STS-3 Gene X STS-4 A. Explain the general strategy of positional cloning. B. Is you applies the approach of positional cloning to clone gene X, where would you begin? As you progressed in your cloning efforts, how would you know if you were walking toward or away from gene X? C. How would you know you had reached gene X? (Keep in mind that gene X exists as a normal allele and a disease-causing allele).

A. Positional cloning is the process of cloning a gene based on its mapped position along a chromosome. It is a successful approach in cloning of many human genes, particularly those that cause genetic diseases when mutated. Commonly use chromosome walking as a method of positional cloning. You begin with a clone that has a marker that is known to map relatively close to the gene of interest. A piece of DNA at the end of the insert is subcloned and then used in a Southern blot to identify an adjacent clone in a cosmid DNA library. This is the first "step." The end of this clone is subcloned to make the next step. And so on. Eventually, after many steps, you will arrive at your gene of interest. B. You would begin at STS-3 because it would require the least amount of steps to get to gene X. If you walk a few steps and get to STS-2 then you would know that you went in the wrong direction. C. You know you reach the gene of interest when spot where the DNA sequences differ between the unaffected and the affected individuals (mutant and normal), which may be within the gene of interest. You can confirm this by sequencing the region from several unaffected and affected individuals to be certain the change in DNA sequencing is correlated with the disease.

Describe the technique of in situ hybridization. Explain how it can be used to map genes-

ANS: In situ hybridization, a probe is used to detect the location of the gene of interest within a set of chromosomes. This is done in a series of steps. First, the cells are treated with agents that swell and fixes them onto a slid. The chromosomal DNA is then denatured and single-stranded probes that have biotin in them are added. To the biotin, avidin, which is fluorescently labeled, is added and bind to the biotin (probe). Thus, the genes are viewed by a fluorescent microscope. If more than one probe is used the order of genes along a chromosome can be determined.

E15: Compared with a conventional plasmid, what additional sequences are required in a YAC vector so it can behave like an artificial chromosome? Describe the importance of each required sequence.

ANS: YACs will require two telomere sequences, and an ARS sequence. The telomeres are needed to prevent shortening of the artificial chromosome at the end. The centromere sequence is needed for proper segregation of the artificial chromosome during meiosis and mitosis. The ARS sequence Is the yeast equivalent of an origin of replication, which is needed so that YAC DNA can be replicated.

3. Chromosomal walking is a type of _______ in which a researcher begins at a specific site on a chromosome and analyzes _______ until the gene of interest is reached. A. DNA sequencing, a series of subclones B. positional cloning, a series of subclones C. DNA sequencing, bands on a gel D. positional cloning, bands on a gel

B. positional cloning, a series of subclones

E4: The cells from a malignant tumor were subjected to in situ hybridization using a probe that recognizes a unique sequence on chromosome 14. The probe was detected only once in each of these cells. Explain these results and speculate on their significance with regard to the malignant characteristics of these cells.

-A normal cell contains two copies of chromosome 14, thus, indicating that in situ hybridization the probe would be detected two times in each cell. The results of this show that the probe was detected only once. This is due to the fact that the malignant cancerous cell could have got rid of the second chromosome 14 in these cells due to uncontrollable cell growth-

E13: In the Human Genome Project, researchers have collected linkage data from many crosses in which the male was heterozygous for markers and many crosses where the female was heterozygous for makers. The distance between the same two markers, computed in map units or centiMorgans, is different between males and females. In other works, the linkage maps for human males and females are not the same. Propose an explanation for this discrepancy. Do you think the sizes of chromosomes (excluding the Y chromosome) in human males and females are different? How could physical mapping resolve this discrepancy?

-Answer- An explanation is that the rate of recombination between homologous chromosomes is different duringoogenesis compared to spermatogenesis. Physical mapping measures the actual distance (in bp) betweenmarkers. The physical mapping of chromosomes in males and females reveals that they are the samelengths. Therefore, the sizes of chromosomes in males and females are the same. The differences obtained in linkage maps are due to differences in the rates of recombination during oogenesis versusspermatogenesis-

RFLP:

- Restriction fragment length polymorphism - A site in a genome where the distance between two restriction sites varies among different individuals - These sites are identified by restriction enzyme digestion of chromosomal DNA and the use of Southern blotting

1. Distinguish between RFLP, AFLP, STR, SNP and STS markers.

-RFLP markers are restriction fragment length polymorphism in which a site in a genome where the distance between two restriction sites varies among different individuals. These sites are identified by restriction enzyme digestion of chromosomal DNA and the use of Southern blotting. -AFLP are amplified restriction fragment length polymorphism. They are the same as an RFLLP except that the site is amplified via PCR instead of isolating the chromosomal DNA. AFLPs, Microsatellites, and SNPs can provide sequence-tagged sites within a genome. -STRs or microsatellites are a site in the genome that contains many short sequences that are repeated many times in a row. The total length is usually in the size range of 50-200 bp, and their lengths may be polymorphic within a population. They are isolated via PCR. Microsatellites are also called short tandem repeats (STRs) and simple sequence repeats (SSRs). -SNPs are single-nucleotide polymorphism and is the site in the genome where a single nucleotide is polymorphic among different individuals. These sites occur commonly in all genomes, and they are gaining greater use in the mapping of disease-causing alleles and in the mapping of genes that contribute to quantitative traits that are valuable in agriculture. -STSs are sequence-tagged sites and is a general term to describe any molecular marker that is found at a unique site in the genome and is amplified by PCR

E7: A researcher is interested in a gene found on human chromosome 21. Describe the expected results of a FISH experiment using a probe that is complementary to this gene. How many spots would you see if the probe was used on a sample from an individual with 46 chromosomes versus an individual with Down Syndrome.

-In an unaffected individual, there would be two spots observed from the two copies of chromosome 21 in the cell. In an individual with down syndrome, three spots would be observed because that person has three copies of chromosome 21-

1. Distinguish between the following types of maps: cytogenetic, linkage, and physical. What units are used to describe locations/distances in each of these types of maps?

Cytogenetic mapping is mapping aimed at determining the locations of specific sequences, such as gene sequences, within chromosomes that are viewed microscopically. Staining is used to describe the locations/distances in this mapping. Stained chromosomes has a characteristic banding pattern and genes are mapped cytogenetically relative to a band location. Linkage mapping uses the frequency of genetic recombination between different genes to determine their relative spacing and order along a chromosome. In Eukaryotes, linkage mapping involves crosses among organisms that are heterozygous for two or more genes. The locations/distances in this mapping are determined by the number of recombinant offspring, computed in map units. Physical mapping uses DNA-cloning-techniques to determine the location of and distance between genes and other DNA regions. The distances are computed as the number of base pairs between genes.