Chapter 22

What are the structural components of a YAC vector?

- 2 TEL - CEN - ORI - ARS - selectable marker - a site where a large piece of chromosomal DNA can be inserted

In a typical walking experiment, each clone might have an average insert size of:

- 50,000 bp - it takes about 20 walking steps to reach the gene of interest

Physical mapping

- DNA-cloning-techniques are used to determine the location of and distance between genes and other DNA regions - the distances are computed as the number of base pairs bw genes

Fluorescence in situ hybridization (FISH)

- most common method of in situ hybridization that uses fluorescently labeled DNA probes

What is a DNA library and how is it obtained?

A DNA library contains a collection of recombinant vectors in which each vector contains a particular fragment of chromosomal DNA It's obtained by digesting chromosomal DNA into small fragments

What is a contig?

A contig is a series of clones that have overlapping fragments of DNA that span a region of chromosome or an entire chromosome.

What is a genetic map?

A genetic map is a chart that describes the order and relative distances between genes and other sites along a chromosome.

For each of the following, decide if 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, this is only one chromosome in the genome. 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

Answer: A. Cytogenetic mapping B. Linkage mapping C. Physical mapping D. Cytogenetic mapping E. Linkage mapping F. Physical mapping

What causes microsatellites to be polymorphic?

Microsatellites are polymorphic when the number of repeat sequences varies

What's a contig?

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

The technique of fluorescence in situ hybridization involves the use of a ______ that hybridizes to a _____

fluorescent probe specific site on an intact chromosome

It is easier to identify many molecular markers w/in a given species' genome rather than identifying many allelic differences among individuals:

for this reason geneticists increasingly turned to molecular markers as points of reference along genetic maps

To map the distance b/w molecular markers via crosses, the marker must be:

polymorphic

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

positional cloning a series of subclones

a molecular marker is a _____ found at a specific site on a chromosome that has properties that allow it to be ______

segment of DNA uniquely identified using molecular tools

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?

- If the sample was from an unaffected individual, two spots (one on each copy of chromosome 21) would be observed. - Three spots would be observed if the sample was from a person with Down syndrome, because the person has three copies of chromosome 21.

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

- In situ hybridization is a cytological method of mapping. - The technique is described in Figure 22.2. A probe that is complementary to a chromosomal sequence is used to locate the gene microscopically within a mixture of many different chromosomes. - Therefore, it can be used to cytologically map the location of a gene sequence. -When more than one probe is used, the order of genes along a particular chromosome can be determined.

Linkage mapping

- use the frequency of genetic recombination bw 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 number of recombinant offspring provides a relative measure of the distance bw genes, which is computed in map units (mu).

How can a particular microsatellite be amplified by PCR?

- using primers complementary to the unique DNA sequences that flank a specific (CA)n region - the PCR primers copy only a particular microsatellite, but not the thousands of others that are interspersed throughout the genome

Polymorphic

- w/ alleles, molecular markers may be polymorphic: w/in a population, they may vary from individual to individual

YACs

- yeast artificial chromosome - can be several hundred thousand to 2 million bp in length - a few hundred YACs are sufficient to create a contig w/ fragments that span the entire length of the chromosome

The technique of fluorescence in situ hybridization (FISH) *figure 22.2 caption description*

1) the 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; these are sister chromatids 2) therefore, each x-shaped chromosome actually contains 2 copies of a particular gene 3) bc the sister chromatids are identical, a probe that recognizes one sister chromatid will also bind to the other

Steps of FISH:

1) treat cells with agents that make them swell and fixes them onto slides 2) denature chromosomal DNA 3) Add single-stranded probes that have biotin incorporated into them 4) Add fluorescently labeled avidin that binds to biotin 5) view w/ fluorescence microscope

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. E. An STS is a molecular marker.

A. False, they do not have to carry genes. B. True. C. False, the marker may not carry a gene that affects phenotype. D. True. E. True.

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

Answer: - A contig is a collection of clones that contain overlapping segments of DNA that span a particular region of a chromosome. - To determine if two clones are overlapping, one could conduct a Southern blotting experiment. - In this approach, one of the clones is used as a probe. If it is overlapping with the second clone, it will bind to it in a Southern blot. - Therefore, the second clone is run on a gel and the first clone is used as a probe. - If the band corresponding to the second clone is labeled, this means that the two clones are overlapping

Why is the technique of chromosome walking useful?

Chromosome walking is used to identify a gene that has already been mapped to a site along a chromosome.

*Figure 22.1*

Figure 22.1 compares genetic maps that show the loci for two X-linked genes, sc (scute, a gene affecting bristle morphology) and w (a gene affecting eye color), in Drosophila melanogaster. In the cytogenetic map at the top, the sc gene is located at band 1A8, and the w gene is located at band 3B6. In the linkage map in the middle, genetic crosses indicate that the two genes are approximately 1.5 map units (mu) apart. The physical map at the bottom shows that the two genes are approximately 2.4 x 10^6 bp apart along the X chromosome.

Which blotting method was originally used to determine if 2 different clones contained an overlapping region?

Southern blotting

CEN

The YAC vector has 1 centromere (CEN)

TEL

The YAC vector has 2 telomeres (TEL)

ORI

The YAC vector has a bacterial origin of replication (ORI)

ARS

The YAC vector has a yeast origin of replication (ARS) - autonomous replication sequence

Why does the probe bind to a specific site on a chromosome?

The probe binds only to a specific site that has a complementary sequence

In an in situ hybridization experiment, what is the relationship between the base sequence of the probe DNA and the site on the chromosomal DNA where the probe binds?

They are complementary to each other.

Molecular markers

a segment of DNA found at a specific site along a chromosome w/ properties that enable it to be uniquely recognized using molecular tools such as polymerase chain reaction [PCR] and gel electrophoresis

How large is the human genome?

approximately 3 billion bp in length

Explain the use of molecular markers in mapping studies:

as an alternative to relying on allelic differences b/w genes, geneticists have realized that regions of DNA that don't encode genes can be used as markers along a chromosome

What can be concluded by two bands of different length observed using PCR primers?

b/c the same site on the two homologous chromosomes contains different numbers of repeat sequences

Positional cloning can be achieved by:

chromosome walking

In situ hybridization localizes:

genes along particular chromosomes

pedigree analysis involving STS, such as polymorphic microsatellites, enables:

identification of the location of disease causing alleles

Linkage mapping can use:

molecular markers

Linkage mapping commonly uses:

molecular markers called microsatellites

What is an example of a molecular marker?

- RFLP - microsatellite - single nucleotide polymorphism

Locus/loci

- refers to the site within a genetic map where a specific gene or other DNA segment is found

A physical map of a chromosome is constructed by:

creating a contiguous series of subclones that span a chromosome

What type of mapping relies on microscopy?

cytogenetic mapping

In general, most plasmid and viral vectors can accommodate:

inserts only a few thousand to tens of thousands of nucleotides in length

What happens if a plasmid or viral vector has a DNA insert that is too large?

it will have difficulty with DNA replication and is likely to suffer deletions in the insert

Researchers can make genetic maps of:

large eukaryotic chromosomes

How is a highly refined map of a genome made?

many different polymorphic sites must be identified and their transmission followed from parent to offspring over many generations

By identifying regions w/in clones that overlap w/ each other:

member of the library can be organized according to their actual locations along a chromosome

Define mapping:

the experimental process of determining the relative locations of genes or other segments of DNA along individual chromosomes

A goal of cytogenetic mapping is to determine:

the location of a gene along an intact chromosome

the distances b/w linked molecular markers can be determined from:

the outcomes of crosses

Subcloning

the procedure of making smaller DNA clones from a larger one

Proteomics

- determine the roles of all cellular proteins to produce the characteristics of particular cell types and the traits of complete organisms

Cytogenetic mapping

- also called cytological mapping - aimed at determining the locations of specific sequences, such as gene sequences, within chromosomes that are viewed microscopically - when stained, each chromosome of a given species has a characteristic banding pattern, and genes are mapped cryogenically relative to a band location

AFLP

- amplified restriction fragment length polymorphism - the same as an RFLP except that the site is simplified via PCR instead of isolating the chromosomal DNA

BACs

- bacterial artificial cloning - developed from bacterial F factors - typically can contain inserts up to 300,00 bp and sometimes larger - easier to use than YAC bc the DNA is inserted into a circular molecule and transformed into E.coli - more commonly used than YACs for the cloning of large DNA fragments

By following the transmission of many polymorphic markers w/in large family pedigrees, it may be possible to:

- determine that particular markers are found in people who carry specific disease-causing alleles - after the identification of a closely linked marker, a disease-causing allele can be identified using a technique called chromosome walking

Genome sequencing projects

- research endeavors w/ the ultimate goal of determining the sequence of DNA bases of the entire genome of a given species - involves many participants including scientists who isolate DNA and perform DNA-sequencing rxns, and theoreticians who gather the DNA sequence information and assemble it into a long DNA sequence for each chromosome

Microsatellites that have length polymorphisms allow:

- researchers to follow their transmission from parent to offspring - PCR amplification of particular microsatellites provides a strategy in the genetic analysis of human pedigrees

What is the ultimate goal of physical mapping procedures?

To obtain a complete contig for each type of chromosome w/in a full set - for humans, a complete physical map requires a contig for each of the 22 autosomes and for the X and Y chromosomes

YAC, BAC, and PAC vectors are used to:

clone large segments of DNA

E18. What is an STS? How are STSs generated experimentally? What are the uses of STSs? Explain how a microsatellite can be a polymorphic STS.

*Answer:* A sequence-tagged site (STS) is a segment of DNA, usually quite short (e.g., 100 to 400 bp in length), that serves as a unique site in the genome. STSs are identified using primers in a PCR reaction. - STSs serve as molecular markers in genetic mapping studies. Sometimes the region within an STS may contain a microsatellite. - A microsatellite is a short DNA segment that is variable in length, usually due to a short repeating sequence. - When a microsatellite is within an STS, the length of the STS will vary among different individuals or even the same individual may be heterozygous for the STS. - This makes the STS polymorphic. Polymorphic STSs can be used in linkage analysis, because their transmission can be followed in family pedigrees and through crosses of experimental organisms.

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.

- Because normal cells contain two copies of chromosome 14, one would expect that a probe would bind to complementary DNA sequences on both of these chromosomes. - If a probe recognized only one of two chromosomes, this means that one of the copies of chromosome 14 has been lost, or it has suffered a deletion in the region where the probe binds. - With regard to cancer, the loss of this genetic material may be related to the uncontrollable cell growth.

Positional cloning

- a gene is cloned based on its mapped position along a chromosome - successful in the cloning of many human genes, particularly those that cause genetic diseases when mutated

Genetic Map

- also called chromosome map - is a chart that describes the relative locations of genes or other DNA segments along a chromosome

The number of steps required to reach the gene of interest depends on:

- the distance bw the starting and ending points and on the sizes of the DNA inserts in the library - if the 2 points are 1 mu apart, they are expected to be approximately 1 million bp apart

What is the most common microsatellite in humans?

- the most common microsatellite encountered in humans is a dinucleotide sequence (CA)n, where *n* ranges from 5 to more than 50 - this dinucleotide sequence can be tandemly repeated 5-50 or more times - the (CA)n microsatellite is found, on avg, about every 10,000 bases in the human genome

Genome

- the total genetic composition of an organism or species - the nuclear genome of humans is composed of 22 different autosomes and an x chromosome and in males a Y chromosome - humans also have a mitochondrial genome composed of a single circular chromosome

Sequence tagged site [STS]

- when DNA is collected from a haploid cell, an STS produces only a single band on a gel - in a diploid species, an individual has 2 copies of a given STS - when and STS contains a microsatellite, the 2 PCR products may be identical and result in a single band on a gel if the region is the same length in both copies (i.e., if the individual is homozygous for the microsatellite) - HOWEVER: if an individual has 2 copies that differ in the number of repeats in the microsatellite sequence (i.e., if the individual is heterozygous for the microsatellite), the 2 PCR products obtained will be different in length

The frequency of crossing over provides:

- a measure of the map distance b/w different microsatellites - this approach can help obtain a finely detailed linkage map of the human chromosomes w/out having to depend on alleles of closely linked genes that affect phenotype

Contig

- a series of clones that contain contiguous (touching), overlapping pieces of chromosomal DNA - represents a physical map of a chromosome - different experimental strategies can be used to align the members of a contig

STR/microsatellites

- 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 w/in a population - they are isolated via PCR - micro satellites are also called short tandem repeats [STRs] and simple sequence repeats [SSRs]

Chromosome walking

- one method of positional cloning - initiation begins with the known position from mapping studies of a gene relative to a marker - this provides a starting point to molecularly "walk" toward the gene of interest - to begin chromosome walking, a cloned DNA fragment that contains a previously cloned gene, and flanking sequences can be used as a starting point - To walk from the previously cloned gene to the gene of interest, a series of library screening methods are followed - a small piece of DNA from the first cosmic vector containing the previously cloned gene is inserted into another vector (subcloning) - the sub cloned DNA is labeled and used as a probe to screen a cosmic library which enables the researcher to identify a second clone that extends into the region that is closer to the gene of interest - a subclone from this second clone is then used to screen the library a second time which allows the identification of a clone that is even closer to the gene of interest - this repeated pattern of subcloning and library screening is used to reach the gene of interest

RFLP

- restriction fragment length polymorphism - a site in a genome where the distance b/w 2 restriction sites varies among different individuals - these sites are identified by restriction enzyme digestion of chromosomal DNA and the use of southern blotting

STS

- sequence tagged site - this is a general term to describe any molecular marker that is found at a unique site in the genome and is amplified by PCR - AFLPs, micro-satellites, and SNPs can provide sequence tagged sites w/in a genome

E19. A human gene, which we will call gene X, is located on chromosome 11 and is found as a normal allele and a recessive diseasecausing allele. The location of gene X has been approximated on the map shown here that contains four STSs, labeled STS-1, STS-2, STS-3, and STS-4. A. Explain the general strategy of positional cloning. B. If you applied 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.)

Answer: [A.] The general strategy is shown in Figure 22.12. The researcher begins at a certain location and then walks toward the gene of interest. -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.] In this example, you would begin at STS-3. -If you walked a few steps and happened upon STS-2, you would know that you were walking in the wrong direction. [C.] This is a difficult aspect of chromosome walking. Basically, you would walk toward gene X using DNA from a normal individual and DNA from an individual with a mutant gene X. -When you have found a site where the sequences are different between the normal and mutant individual, you may have found gene X. -You would eventually have to confirm this by analyzing the DNA sequence of this region and determining that it encodes a functional gene.

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.

Answer: - Besides a selectable marker and an origin that will replicate in E. coli, YAC vectors also require two telomere sequences, a centromere sequence, and an ARS sequence. - The telomeres are needed to prevent the shortening of the artificial chromosome from the ends. The centromere sequence is needed for the 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 the YAC DNA can be replicated.

SNP

- single nucleotide polymorphism - a 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

method of in situ hybridization:

- widely used to cytogenetically map the locations of genes or other DNA sequences within large eukaryotic chromosomes - in situ: "in place" indicates that the procedure it conducted on chromosomes that are being held in place- adhered to a surface 1) use a probe to detect the location of the gene w/in a set of chromosomes; if the gene of interest has been cloned previously, the DNA of the cloned gene can be used as a probe 2) b/c a DNA strand from a cloned gene, which is a very small piece of DNA relative to a chromosome, hybridizes only to its complementary sequence on a particular chromosome - this technique provides a way of localizing the gene of interest

S2. An RFLP marker is 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 5000 bp. Approximately how many steps will it take to get there?

Answer: - Each step is only 50,000 bp (i.e., 55,000 minus 5000), 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.

In large eukaryotic genomes, beginning with vectors that can accept chromosomal DNA inserts of very large size is preferred b/c:

by having large size inserts, a contig is more easily constructed and aligned bc fewer recombinant vectors are needed

How do researchers know when they have reached a gene of interest?

- in the case of a gene that causes a disease when mutant, researcher conduct walking steps on DNA from both an unaffected and an affected individual - each set of clones is subjected to DNA sequencing, and those DNA sequences are compared w/ each other - when a spot is reached where the DNA sequences differ bw the unaffected and the affected individual, such a site may be within the gene of interest - this site is confirmed by sequencing the region from several unaffected and affected individuals to be certain the change in DNA sequence is correlated with the disease

Why are molecular markers useful?

- one key reason is that molecular markers can be used to determine the approximate location of an unknown gene that causes human disease - following transmission patterns of polymorphic molecular markers in family pedigrees to identify genes that, when they are muted, cause human disease - the discovery of a particular marker in those who have the disease can indicate that the marker is close to the disease-causing allele - genetic maps w/ a large number of markers are used by evolutionary biologists to determine patterns of genetic variation w/in a species and the evolutionary relatedness of different species

Microscopically, eukaryotic chromosomes can be distinguished from one another by:

- size - centromeric location - banding patterns : obtained by treating chromosomes with particular dyes, a discrete banding pattern is obtained

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 library. C. Subclone BAC fragments to make a cosmid library. D. Subclone cosmid fragments for DNA sequencing.

Answer: The proper order is A, C, D, B. 1. Clone large fragments of DNA to make a BAC library. 2. Subclone BAC fragments to make a cosmid library. 3. Subclone cosmid fragments for DNA sequencing. 4. Determine the DNA sequence of subclones from a cosmid library.

[S3.] Does a molecular marker 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.

[Answer:] A molecular marker does not have to be polymorphic to be useful in physical or cytological mapping studies. - Many sequencetagged sites (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.* - *Polymorphic molecular markers 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.

Map correlations:

correlations among cytogenetic, linkage, and physical maps often vary from species to species and from one region of the chromosome to another

What is the goal of linkage studies?

- to follow the transmission of many different microsatellites to determine those that are linked along the same chromosome vs those that aren't - those that aren't linked will independently assort from generation to generation - those that are linked tend to be transmitted together to the same offspring - in large pedigrees, it's possible to identify cases in which linked microsatellites have segregated due to crossing over

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

Answer: - BAC cloning vectors have the replication properties of a bacterial chromosome and the cloning properties of a plasmid. - To replicate like a chromosome, the BAC vector contains an origin of replication from an F factor. Therefore, in a bacterial cell, a BAC can behave as a chromosome. - Like a plasmid, BACs also contain selectable markers and convenient cloning sites for the insertion of large segments of DNA. - The primary advantage is the ability to clone very large pieces of DNA.

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?

- The term fixing refers to procedures that chemically freeze cells and prevent degradation. After fixation has occurred, the contents within the cells do not change their morphology. In a sense, they are frozen in place. - For a FISH experiment, this keeps all the chromosomes within one cell in the vicinity of each other; they cannot float around the slide and get mixed up with chromosomes from other cells. - Therefore, when we see a group of chromosomes in a FISH experiment, this group of chromosomes comes from a single cell. - It is necessary to denature the chromosomal DNA so that the probe can bind to it. - The probe is a segment of DNA that is complementary to the DNA of interest. - The strands of chromosomal DNA must be separated (i.e., denatured) so that the probe can bind to complementary sequences.

*Answer:* Keep in mind that mature sperm are haploid, so they have only one copy of STS-1 and one copy of STS-2. A. If we look at the 40 lanes, most of the lanes (i.e., 36 of them) have either the 234-bp and 423-bp STSs or the 198-bp and 322-bp ones. This is the arrangement of STSs in this male. One chromosome has STS-1 that is 234 bp and STS-2 that is 423 bp, and the homologous chromosome has STS-1 that is 198 bp and STS-2 that is 322 bp. B. There are four recombinant sperm, shown in lanes 15, 22, 25, and 38. Map distance = 4/40 × 100 = 10.0 mu [Note:] This is a relatively easy experiment compared with a pedigree analysis, which would involve contacting lots of relatives and collecting samples from each of them.

[S1.] The distance between two molecular markers that are linked along the same chromosome can be determined by analyzing the outcome of crosses. -This can be done in humans by analyzing the members of a pedigree. -However, the accuracy of linkage mapping in human pedigrees is fairly limited because the number of people in most families is relatively small. -As an alternative, researchers can analyze a population of sperm, produced from a single male, and compute linkage distance in this manner. -As an example, let's suppose a male is heterozygous for two polymorphic STSs. STS-1 exists in two sizes: 234 bp and 198 bp. STS-2 also exists in two sizes: 423 bp and 322 bp. -A sample of sperm was collected from this man, and individual sperm were placed into 40 separate tubes. In other words, there was one sperm in each tube. -Believe it or not, PCR is sensitive enough to allow analysis of DNA in a single sperm! -Into each of the 40 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 two STSs in this individual? B. What is the map distance between STS-1 and STS-2?

Genomics

- molecular analysis of the entire genome of a species - genome analysis is a molecular dissection process applied to a complete set of chromosomes - segments of chromosomes are analyzed in progressively smaller pieces, the locations of which are known on the intact chromosomes - this is the mapping phase of genome analysis: the mapping of the genome ultimately progresses to the determination of the complete DNA sequence for all of a species' chromosomes