Module 3 Exam

Name and describe three different kinds of bacterial cloning vectors.

(1) plasmid: contain a multi-cloning site, origin of replication, and a selectable marker; can carry ~20-25Kb of foreign DNA (2) phage: a virus containing the DNA of interest infects bacteria; is more efficient than plasmid transformation and can carry ~25Kb of foreign DNA (3) cosmid: type of hybrid plasmid that contains plasmid sequences plus the COS sequences from phage for capsid packaging for phage transduction; cosmids form colonies, not plaques; can carry up to ~45Kb of foreign DNA (4) bacterial artificial chromosome or BA: a vector based on the bacterial F-plasmid (fertility plasmid), making its introduction in bacteria more stable than plasmids; typical foreign DNA insert size is in the hundreds of Kb

Write the letter all of the following statements that are NOT true.

(B) Antibodies are used for Northern blot analysis.

List at least three different kinds of bacterial cloning vectors.

-Cosmids -Bacterial artificial chromosomes (BAC) -Expression vector

1) What enzyme is required for PCR? State the specific name of the enzyme, not just the type of enzyme.

Taq polymerase

Match this term: PCR

Taq polymerase

The ___________ operon is typically characterized by the following two terms: negative control and repressible.

tryptophan

Match each number with the closest type of DNA: a. moderately repetitive DNA b. highly repetitive DNA c. short interspersed elements d. long interspersed elements e. unique-sequence DNA Reference: Ref 11-1 gene-encoding sequence Enter only the letter of your choice.

unique-sequence DNA

MC: The difference between a genetic screening experiment and a selection experiment is that a screening experiment involves ________, whereas a selection experiment creates conditions that ________ irrelevant organisms.

visual examination, eliminate

Describe the standard PCR method (in sufficient detail) and how this process is able to produce clones in a "cell-free" system.

Target DNA is heated and denatured giving two single strands. The DNA solution is then cooled so the primers can attach to the template strands. The solution is heated again so DNA polymerase can synthesize new DNA strands using Taq polymerase. The cycle is then repeated. All of the cellular machinery can run in an artificial environment so it does not require a host cell.

Which of the following statements about manual Sanger sequencing is true?

The DNA sequence obtained is complementary to the template strand.

PCR: 2) Apart from the enzyme, what 3 DNA molecules are required (give the technical names by which they are called)? What must be true about the sequences of these molecules? Note that dNTPs are individual nucleotides, not DNA molecules.

The complement, single DNA strand and 2 short oligonucleotide primers are required to flank each side of the target sequence. The sequence of the primers and complement DNA strand must be complementary to one another, so the primers can bind to the DNA.

Intron frequency varies considerably among eukaryotes. Provide a general comparison of intron frequencies in yeast and humans. What about intron size?

The entire yeast genome has only about 240 introns, whereas some single genes in humans contain over 100 introns. In general, smaller genomes have smaller intron size in addition to lower intron number.

In general, mutation rates in humans occur in the range of ________ (frequency) per gamete per generation.

10^-5 to 10^-6

MC: You are doing an experiment to characterize a 3000bp clone using two different restriction enzymes. Enzyme 1 (E1) produces 2 fragments in a single digest of 1400 and 1600bp. Enzyme 2 (E2) also produces 2 fragments of 1400 and 1600bp. When a double digest using both of these enzymes is done, it results in 2 fragments of 1400bp and 200bp. Based on this data, choose the correct restriction map from the choices given below.

1400,E1,200,E2,1400

You determine that you have only three copies left of an important DNA fragment, so you decide to amplify it. Using flanking primers, how many PCR cycles would you have to run to generate over one billion (10^9) copies of the fragment?

32^x=110^9 x=28.3 29 times in order to get over one billion copies

The haploid human genome contains about 3 × 109nucleotides. On average, how many DNA fragments would be produced if this DNA was digested with restriction enzyme PstI (a 6-base cutter)? RsaI (a 4-base cutter)? How often would an 8-base cutter cleave?

4 base cutter = (1/4)^4 = 1/256bp gets cut (3x10^9)/256 = 11,718,750 cuts 6 base cutter = (1/4)^6 = 2.44e-4 ~ 1/5000bp gets cut (3e9/5000) = 600,000 cuts 8 base cutter = (1/4)^8 = 1e-5 ~1/100,000 gets cut (3e9/100000) = 30,000 cuts

Restriction endonucleases are typically used to clone genes. What factors determine the sites at which these endonucleases will cleave DNA? What characteristics do these sites tend to have?

Each RE will cleave at a specific sequence. These sequences tend to be short (4-8 bp), and tend to be palindromic (e.g., GAATTC).

We have looked at the cloning experiments involved in producing Snuppy. Describe the specific technique that was used and how the results demonstrated that Snuppy was in fact a clone of the donor Afghan hound.

Microsatellite analysis was used to show that Snuppy was a clone. Microsatellite loci are highly variable loci that contain a large number of DNA repeats (eg. 2, 3, or 4 nulceotides in length) at the population level. However, an individual can only have 2 of these alleles at any one microsatellite locus. By comparing the alleles that Snuppy had at 8 different microsatellite loci with those alleles that the donor Afghan hound and the surrogate mother had, it was shown that Snuppy had exactly all of the same alleles as the Afghan hound, proving that Snuppy was a clone of the donor Afghan.

You determine that you have only three copies left of an important DNA fragment, so you decide to amplify it. Using flanking primers, how many PCR cycles would you have to run to generate over one billion (109) copies of the fragment?

Accept anywhere from 28 to 30 cycles as a correct answer or the following equation 3 x (2^n) => than 1 billion)

MC: A section of a genome is cut with three enzymes: A, B, and C. Cutting with A and B yields a 10-kb fragment. Cutting with B and C yields a 2-kb fragment. What is the expected result from a digest with A and C, if the C site lies in between the A and B sites?

An 8-kb fragment

One of the dominant features of the immune system is the capacity to generate new cells that contain different combinations of antibodies. Because there are billions of such combinations it is impossible that each combination is coded by a separate gene. Explain in as much detail as you can how such diversity is accomplished in the case of the light chain of a typical antibody.

An antibody is a Y-shaped molecule that contains 4 chains (2 heavy and 2 light). There are two types of light chains and 5 types of heavy chains. Different combinations of chains create different types of antibody classes. Each mature B cells makes one type of light chain and one type of heavy chain. The light chain genes have many different regions.

What is a transgenic organism?

An organism that has been permanently altered by the addition of a DNA sequence to its genome.

MC: Of the DNA sequences below, which would probably be the harder to determine?

CGATATATAT... The repetitive region in A would make it harder to determine even though it is shorter.

Rank from "roughest" → "fine detail" the amount of resolution allowed by the following methods of mapping:

Cytogenetic, linkage, restriction, sequence

MC: During gel electrophoresis, __ will migrate more rapidly than __.

D. small DNA fragments C. large DNA fragments

Which of the following are the important proteins needed for cloning a eukaryotic gene into a bacterial plasmid?

DNA ligase restriction enzymes specific for the target genes

Match this term: real-time PCR

DNA quantification

Explain why the genetic map distance between two genes on the same chromosome may be inconsistent with the physical map distance. E.g., for three loci A, B, and C, on the same chromosome, explain why the genetic distance might be A-[20 centimorgans]-B-[20 cM]-C, while the physical distance might be A-[200 kilobases]-B-[100 kb]-C.

Different regions of the chromosome will be more prone to recombination than others.

Describe what is meant by the term forward genetics.

Forward genetics involves the isolation of mutants that show differences in a particular phenotype of interest. Mutant isolation is usually followed by an analysis of gene pathways through epistasis and/or complementation analyses. Then the gene is usually mapped and cloned and studied by a variety of molecular and developmental approaches.

List four uses of PCR.

Gene cloning, detect the presence of DNA sequence in a sample, detect the presence of viruses in blood samples, determine the amount of starting nucleic acid.

Crossing over is often reduced around centromeric regions of chromosomes. If you were trying to construct a genetic map of two linked marker loci in this region, what result might you obtain and why? How would the genetic map correspond to the physical map?

Genes mapped based on recombination will appear to be very close together in centromeric regions due to low rates of recombination. Distances between the same genes on the physical map may be much greater when compared to other regions of the chromosome.

Explain why genetic and physical map distances may differ in relative distances between two genes on a chromosome.

Genetic maps are measured in percent recombination (map units). This does not always accurately correspond to physical distances because they are based of rates of crossing over. Physical maps measure distances in number of base pairs which makes it more accurate.

MC: List two especially useful characteristics of cloning vectors.

High copy number and antibiotic resistance genes

In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling in the second step?

In PCR, the initial high temperature serves to denature the double stranded DNA into single stranded DNA. Cooling in the second step allows for the annealing or hybridization of the primer to the DNA strand, flanking either side of the target DNA.

Nucleic acid blotting is commonly used in molecular biology. Two types, Southern blots and northern blots, involve gel electrophoresis and a filter, which holds the nucleic acid. Briefly describe the procedure of "blotting" in this context and differentiate between Southern and northern blots.

In a Southern blot the DNA to be "probed" is cut with a restriction enzyme(s); then the fragments are separated by gel electrophoresis. Alkali treatment of the gel denatures the DNA, which is then "blotted" onto the filter (nylon or nitrocellulose). A labeled probe (RNA or DNA) is then hybridized to complementary fragments on the filter. In a northern blot, RNA is separated in the gel and "probed" with the labeled DNA.

Cloning reactions are done with DNA that has been cloned by restriction digestion and not by PCR. Using what you know about the way PCR works, why would you not want to use DNA from PCR to create DNA for cloning?

Normally in DNA replication, polymerase makes errors one out of every 1010 nucleotides inserted. (In addition, Taq polymerase used in PCR is less faithful because it does not have a proofreading subunit). Because PCR amplifies from previous sequences if an error is made early on it will be proliferated in the sequence.

What is recombinant DNA technology? What are the safety issues related to recombinant DNA technology?

Recombinant DNA technology refers to the creation of new combinations of DNA molecules that are not normally found in nature. Safety issues generally center around the creation and release (accidental or intentional) of genetically engineered organisms that are a threat to human health or animals and plants in the environment. Many organisms that are "genetically engineered" carry genes for antibiotic resistance.

A map of the distribution of cloned genomic DNA from genomic clone libraries.

Restriction mapping?

MC: Which technique would NOT be used to find a gene for a functional protein in a sequenced region of a genome?

See if a SNP database contains sequences in the region.

Regarding a gene's DNA sequence, what is meant by a sequence motif?

Sequence motifs are short, recurring patterns in DNA that are presumed to have a biological function.

The transcriptome of a genome contains more components than the proteome. Explain why this is true.

The transcriptome contains more components because it is identifying all of the RNA molecules that are transcribed. The proteome contains all of the proteins encoded. Some of the proteins can be coded by the same RNA sequence so there are less components.

Compare the transcriptome of an organism with the proteome. What is described by each?

The transcriptome is the identification of the RNA molecules transcribed from a genome. The proteome is all of the proteins encoded by the genome.

Name at least 2 methods that are used to produce mutations in a forward genetics approach.

UV light, EMS, nitrosguanadine, transposons

Another word for a "DNA chip" (microscopic spots of oligonucleotides bound to glass that can be fluorescently labelled to identify levels of expression).

Microarray

The lungfish Protopterus aethiopicus has a genome 38 times larger than that of humans. Most of the DNA in this species is noncoding repetitive DNA. How could you create a library of clones that would let you compare just the genes in the lungfish to the genes in humans?

You could generate cDNA libraries and compare the transcribed regions of the genome.

BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would NOT be recognized by this enzyme?

[x] b) 3′ TCCTTAAG 5′

Compare the fields of structural, functional, and comparative genomics. What is the purpose of each?

Structural genomics is the organization and sequence of the genetic information within a genome. The purpose of this is to create maps that provide information on locations of genes, molecular markers, and chromosome segments. Functional genomics characterizes what the sequences do. This identifies their function. Comparative genomics compares similarities and differences in gene content, function, and organization among genomes of different organisms.

MC: For a physical map of a chromosome, distances are measured in units of:

base pairs

Match this term: YAC

centromere

Match this term: in situ hybridization

chromosome spread

A PCR technique that fills in small gaps by using the end of a cloned sequence as a primer to amplify into adjacent uncloned fragments.

chromosome walking

Transcription factors are proteins with at least two functional _________.

domains

MC: One of the primary reasons for the necessity of generating a large number of clones in a eukaryotic genomic library is that:

each vector can take up only a relatively small fraction of the eukaryotic DNA

MC: Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot one generally:

hybridizes filter-bound DNA with a DNA probe

Match this term: ß-galactosidase

lacZ

Match this term: cDNA library

mRNA

Match this term: shuttle vector

multiple hosts

What methods are used to produce mutations in a forward genetics approach?

mutagenic agents, x-rays, radiation, chemical mutagens (EMS), transposable elements, UV light.

A gene construct that indicates when transcription occurs because the protein is easily identified (often GUS or GFP).

reporter sequence

MC: Which of the following enzymes is used to make complementary DNA (cDNA) from RNA?

reverse transcriptase

Over the years, sophisticated plasmid vectors have been developed for use in recombinant DNA technology. Describe the useful features that have been introduced in these vectors.

small size to allow large inserts high copy number large numbers of unique restriction sites (polylinkers) variety of selection schemes (pigmented colonies, antibiotic resistance)

What are three key differences between a genomic and a cDNA library?

-cDNA is enriched with fragments from actively transcribed genes cDNA lib. represents only transcribed regions of the genome; all genes equally represented in genomic library while cDNA library reflects the level of expression of a gene in a particular cell type or tissue ; cDNA library contained only sequences found in the mature mRNA - introns are removed -Introns do not interrupt the cloned sequences in cDNA -cDNA contains only sequences that are present in mature mRNA

In a three-point mapping experiment, how many different genotypic classes are expected?

8

One of the dominant features of the immune system is the capacity to generate new cells that contain different combination of antibodies. Because there are billions of combinations it is impossibe for each combination to be encoded by a single gene. Explain in sufficient detail how such diversity is accomplished in the case of the light chain of a typical antibody.

Antibodies are made of segments. There are many different segments, each varying from one another. In the maturation of lymphocytes, segments are combined to form an immunoglobulin gene. The different possible uses of each particular segment create many possible combinations of the segments. The diversity is a direct result of somatic recombination on a single chromosome, where a v segment is moved to a position next to a J segment. After somatic recombination, the B cell is transcribed into mRNA containing one V gene, several J genes, and an additional C gene. This pre mRNA is then processed into a final RNA containing one of each each. This final RNA is then translated into a functional light chain. Each of these stages offer events which accomplish diversity.

In the previous list of cloned fragments, the fragments needed to make the longest possible contig, with the least amount of overlap, are:

BDCF

Molecular biologists rely on many, often sophisticated, techniques to pursue their discipline. One may list ultracentrifugation, electron microscopy, X-ray diffraction, electrophoresis, and computer interfacing as fundamental. Model organisms provide the raw materials for study. List four "organisms" (or organismic groups) often used by molecular biologists and describe a major advantage of each group to the molecular biologist. We might consider these as "model organisms" of the molecular biologist.

Bacteriophage: relatively simple, short generation time. Bacteria: relatively simple, short generation time, simple growth requirements. Yeast: relatively simple for a eukaryote, short generation time, simple growth requirements. Drosophila: relatively simple to culture, extensive genetic and developmental information available, "giant" polytene (salivary gland) chromosomes.

As a model system, what are some of the advantages of the mouse as a model system?

Close evolutionary relationship to humans. Genetically, behaviorally, and physiologically similar to humans. Short generation time. Well adapted to life in the lab. Large litters. Easy to handle.

What is comparative genomics? How does its study contribute to our understanding of genetics?

Comparative genomics is a relatively new field involved in identifying similarities and differences in organization and gene content among the genomes of different organisms. Such studies are important for studying the genetic relatedness of species and for identifying gene families.

PCR: 4) How does this system work to amplify DNA?

Each run of PCR results in double the amount of initial DNA, thus after several runs, the initial target DNA is significantly amplified.

Mario Capecchi, Sir Martin Evans, and Oliver Smithies recently won a Nobel Prize for gene targeting (gene knockouts) in mice. Describe the steps involved in creating a knockout mouse.

Embryonic stem (ES) cells heterozygous for a knockout mutation in a gene of interest (X) and homozygous for a marker gene (here, black coat color) are transplanted into the blastocoel cavity of 4.5-day embryos that are homozygous for an alternate marker (here, white coat color). The early embryos then are implanted into a pseudopregnant female. Some of the resulting progeny are chimeras, indicated by their black and white coats. Chimeric mice then are backcrossed to white mice; black progeny from this mating have ES-derived cells in their germ line. By isolating DNA from a small amount of tail tissue, it is possible to identify black mice heterozygous for the knockout allele. Intercrossing of these black mice produces individuals homozygous for the disrupted allele, that is, knockout mice.

Describe one major difference in the organization or content of prokaryotic and eukaryotic genomes.

Eukaryotic genomes contain repetitive DNA that is largely absent in prokaryotic genomes. -or- Genes are more densely spaced in prokaryotes versus eukaryotes -or- prokaryotic genomes typically encode fewer genes than eukaryotic genomes Other answers are acceptable, provided they are true and make sense.

MC: There are different challenges that exist for sequencing prokaryotic and eukaryotic genomes. Which challenge is correctly paired with the type of genome to which it relates?

Eukaryotic: repetitive DNA

Design an experiment that would allow you to determine if a particular nucleus in a Drosophila embryo is capable of directing development of an entire new fly.

Experiments in support of such genomic equivalence include the following: the observation that chromosome number and structure do not consistently change in different cells of an organism, nuclear transplantation in amphibians, and the presence of genes but no gene products in some tissues (hemoglobin, for example). The recent cloning of Dolly also supports the above. For the Drosophila experiment, one could do nuclear transplantation similar to the classic experiments in amphibians and recent experiments in mammals or transplant a cell from embryos of one genotype to the posterior end of the early embryos of another genotype. The transplanted cells would be reprogrammed into germ cells by the posterior P granules. The transplant can be detected in the next generation by the recognizable different phenotypes due to different genotypes of the donor and recipient.

MC: All of the following are characteristics of the genomics revolution EXCEPT_____

Inability to understand single genes

What is the purpose of the LacZ gene in a plasmid cloning vector?

It is used as a selectable marker. The gene contains a series of unique restriction sites where a fragment of DNA can be inserted and cloned.

What properties must a molecule have to serve as a vector?

It should be able to replicate itself independently, contain a number of unique restriction sites that would enable the insertion of DNA fragments cut with the same enzyme, carry a selectable marker, and be easy to retrieve.

What is meant by the term low gene density? Give an example of an organism with low gene density.

Low gene density is common in eukaryotes in which there may be as few as one gene in 64 kb base pairs, as is the case with a segment of human chromosome 22

Two genes that evolved from the same common ancestral gene, but are now found as homologs in different organisms are called _______.

Orthologs

What is meant by the term pseudogene?

Pseudogenes are nonfunctional versions of genes that resemble other gene sequences but that contain significant nucleotide substitutions, deletions, and duplications that prevent their expression. Pseudogenes are designated by the prefix (psi).

PCR: 3) Name the three basic steps of PCR and describe the molecular processes that occur in each. How is each step induced?

The first step of denaturization is induced by a high temperature, which facilitates the denaturing of double stranded DNA into single stranded DNA. The second step of hybridization/annealing is induced by a lower temp, which allows the primers to bind or anneal to the complement DNA, on either side of the target sequence. The final, third step of elongation is induced by a medium temperature, slightly higher than step 2, which allows the thermodynamically stable Taq polymerase enzyme to elongate the primers by synthesising new DNA.

Of what advantage is it to have a polylinker region (multiple unique restriction sites) embedded in the lacZ component in the pUC series of plasmids?

The increased restriction sites in the polylinker region increase the opportunity for the target DNA fragment to be inserted into the plasmid, and if embedded in the lacZ gene, it increases the liklihood that the gene will be interrupted by the insertion of the DNA. If the lacZ gene is interrupted, B galactosidase is not produced. When plated on X-gal medium, the resulting colonies containing the recombinant DNA will turn white from the inability to cleave X-gal with B galactosidase, as opposed to the blue colonies with a functional, uninterrupted lacZ gene. If the polylinker region is not embedded in the lacZ component, the liklihood that the recombinant DNA will disrupt the lacZ gene is not as high. The advantage creates an increased liklihood of a selective marker that determines the colonies that have recombinant DNA from those with nonrecombinant DNA, based on their color.

You have cut DNA from source A with restriction enzyme #1 and you have cut DNA from source B with restriction enzyme #2. Both of these restriction enzymes leave a 4 base single stranded overhang. You want to ligate these restricted fragments together. What must be true for this to be successful?

The overhangs must be complementary for them to anneal and be sealed by DNA ligase.

Why are telomeres and centromeres particularly difficult to sequence?

They consist of highly repetitive DNA, and so strand slippage issues can confuse the determination of a consensus sequence.

Which of the following statements about ddNTPs is true?

They have a hydrogen at the 3′ carbon of the ribose sugar.

What is the function of dideoxynucleotides in Sanger DNA sequencing?

They stop synthesis at a specific site, so the base at that site can be determined.

MC: What is bioinformatics?

a method that uses very large national and international databases to access and work with sequence information

MC: PCR is:

a technique for amplifying DNA sequences in vitro

Plasmids are important in biotechnology because they are

a vehicle for the insertion of foreign genes into bacteria

Match this term: transgene

foreign DNA

A _____ family is a group of evolutionarily related genes that arose through repeated evolution of an ancestral gene.

gene

The smallest number of clones that represents the entirety of the genome are called what?

gene library

This term refers to the work undertaken by large teams of researchers who, through a concerted effort, clone and sequence the DNA of a particular organism.

gene mapping (Human Genome Project)

This is the study of "genes in their entirety."

genomics

Assume that one conducted a typical cloning experiment using pUC18, transformed an appropriate host bacterial strain (one carrying the lacZ complementing region), and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant pUC18? Explain your answer.

pUC18 is a polylinker, which is characterized by increased multiple resritiction sites. The increased amount of restriction sties would increase the liklihood of the target DNA sequence being inserted into the plasmid. If the DNA sequence is insterted successfully and interrupts the LacZ gene sequence also in the resriction site, the bacteria will be unable to produce B galactosidase, which cleaves X-gal. When x-gal in the medium is not cleaved, the colonies turn white. If DNA was not successfully inserted into host DNA and the LacZ gene can produce B galactosidase, X-gal will be cleaved and the colonies will be blue. Because the white colonies will be the hosts containing the recombinant DNA, and pUC18 increases the likelihood of DNA being inserted, the white colonies will most likely contain the recombinant pUC18. An insert of DNA in the polylinker inactivates the lacZ component, allowing identification of recombinant plasmids under proper genetic and environmental conditions

A map of the order, overlap and orientation of physically isolated pieces of the genome.

physical maps

Match this term: cloning vector

plasmid

Match this term: expression vector

protein?

MC: The set of all proteins encoded by the genome is called the ______

proteome

MC: If a restriction enzyme cuts a circular DNA into three fragments, how many restriction sites are there in the DNA?

three

MC: What is the enzymatic function of restriction enzymes?

to cleave nucleic acids at specific sites