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85 terms

Module 4

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.
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.
See powerpoint slides.
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.
Explain why the greatest diversity of human SNPs is found among African people.
The greatest diversity of human SNPs is found in African people because studies suggest that humans first evolved in Africa. Since Africans have been around the longest they have had the greatest amount of time to create SNPs.
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?
Match this term: cDNA library
What is the purpose of an antibiotic resistance gene in a plasmid cloning vector?
The antibiotic resistant gene provides a selectable marker for cells.
Match this term: cloning vector
Match with definition: Ortholog, Syntheny, Paralog, Homolog.
Homolog: Closely related genes based on sequence and function. Ortholog: Homologus genes of the same locus inherited from a common ancestor. Paralog: Genes related by gene duplication in the genome. Syntheny: Conservation of the same groups of genes in the chromosomes of 2 or more species
Match this term: shuttle vector
multiple hosts
What is a transgenic organism?
An organism that has been permanently altered by the addition of a DNA sequence to its genome.
A _____ family is a group of evolutionarily related genes that arose through repeated evolution of an ancestral gene.
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)
Match this term: real-time PCR
DNA quantification
A gene construct that indicates when transcription occurs because the protein is easily identified (often GUS or GFP).
reporter sequence
A map of the order, overlap and orientation of physically isolated pieces of the genome.
physical maps
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
MC: A set of overlapping DNA fragments that form a contiguous stretch of DNA is called a _________.
MC: 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: 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
MC: Which of the below are not steps in the production of genome sequence maps:
Isolate whole chromosomes
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.
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: PCR is:
a technique for amplifying DNA sequences in vitro
MC: If a restriction enzyme cuts a circular DNA into three fragments, how many restriction sites are there in the DNA?
MC: Plasmids are important in biotechnology because they are:
a vehicle for the insertion of foreign genes into bacteria
MC: All of the following are characteristics of the genomics revolution EXCEPT_____
Inability to understand single genes
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
MC: For a physical map of a chromosome, distances are measured in units of:
base pairs
MC: Which of the following enzymes is used to make complementary DNA (cDNA) from RNA?
reverse transcriptase
MC: Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky?
single stranded complementary tails
MC: Which of the following are the important proteins needed for cloning a eukaryotic gene into a bacterial plasmid?
restriction enzymes specific for the target genes and DNA ligase (Both B and C)
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.
List the three basic components required for a bacterial cloning vector and briefly describe the purpose of each.
-Origin of replication: ensures that the vector is replicated within the cells
-Selectable markers: enable all cells containing the vector to be identified
-Restriction sites: needed so a DNA fragment can be inserted
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.
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.
What are Northern analyses used for? Describe the steps involved in performing a Northern analysis, and describe how levels of gene expression are determined.
MC: During gel electrophoresis, __ will migrate more rapidly than __.
D. small DNA fragments
C. large DNA fragments
Match this term: PCR
Taq polymerase
Match this term: expression vector
Another word for a "DNA chip" (microscopic spots of oligonucleotides bound to glass that can be fluorescently labelled to identify levels of expression).
Write the letter all of the following statements that are NOT true.
(B) Antibodies are used for Northern blot analysis.
Match this term: ß-galactosidase
A map of the distribution of cloned genomic DNA from genomic clone libraries.
Restriction mapping?
In the previous list of cloned fragments, the fragments needed to make the longest possible contig, with the least amount of overlap, are:
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.
Match this term: transgene
foreign DNA
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.
Rank from "roughest" → "fine detail" the amount of resolution allowed by the following methods of mapping:
Cytogenetic, linkage, restriction, sequence
MC: A typical prokaryotic genome has
1 million base pairs of DNA, containing 1000 genes.
MC: A human gene with a disease phenotype is going to be mapped by positional cloning. Which would be the most useful for this task?
An EST database of the human genome
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.
MC: What is the enzymatic function of restriction enzymes?
to cleave nucleic acids at specific sites
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.
MC: Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. What term is given to this advantageous arrangement of restriction sites?
MC: The set of all proteins encoded by the genome is called the ______
MC: What do PCR, reverse transcription, and dideoxy DNA sequencing all have in common?
All produce DNA chains as a product.
MC: A BLAST search is done to:
find similar gene or protein sequences.
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
MC: A principal problem with inserting an unmodified mammalian gene into a bacterial plasmid, and then getting that gene expressed in bacteria, is that
bacteria cannot remove eukaryotic introns.
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
MC: What is bioinformatics?
a method that uses very large national and international databases to access and work with sequence information
The haploid human genome contains about 3 × 109 nucleotides. 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?
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?
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.
In the genetic map of the human genome, one map unit is approximately 850,000 bp. For the genome of the eukaryotic yeast Saccharomyces cerevisiae, one map unit is approximately 3000 bp. What is a map unit, and why is it so different in these two different types of organisms?
A map unit is the distance on genetic maps based on percentage of recombination. This is the rate that the chromosome crosses over. The numbers are different because in general, multicellular eukaryotes have more DNA than simple, single-celled eukaryotes.
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.
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.
Match this term: YAC
Match this term: in situ hybridization
chromosome spread
This is the study of "genes in their entirety."
What methods are used to produce mutations in a forward genetics approach?
mutagenic agents, x-rays, radiation, chemical mutagens (EMS), transposable elements, UV light.
The smallest number of clones that represents the entirety of the genome are called what?
gene library
Two genes that evolved from the same common ancestral gene, but are now found as homologs in different organisms are called _______.
MC: Electrophoresis separates DNA fragments of different sizes, but this technique does not indicate which of the fragments contains the DNA piece of interest. This problem is solved by:
Removing the bands from the gel and hybridizing them with a known strand of DNA complementary to the gene of interest
MC: List two especially useful characteristics of cloning vectors.
High copy number and antibiotic resistance genes
Describe the basic components for a typical plasmid cloning vector system and the reason/use for those plasmid vector components.
-Origin of replication: ensures that the vector is replicated within the cells
-Selectable markers: enable all cells containing the vector to be identified
-Restriction sites: needed so a DNA fragment can be inserted
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.
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.
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?
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.
Insert the knockout gene into embryonic stem cells. Grow transformed ES cells on medium. Select surviving ES cell colonies, test for presence of knockout gene, grow up culture of knockout cells. Insert ES cells into blastocyst. Mate chimera to black mice. Test agouti progeny for presence of knockout gene. Mate siblings to establish homozygous lines.
What are three key differences between a genomic and a cDNA library?
-cDNA is enriched with fragments from actively transcribed genes
-Introns do not interrupt the cloned sequences in cDNA
-cDNA contains only sequences that are present in mature mRNA
List at least three different kinds of bacterial cloning vectors.
-Bacterial artificial chromosomes (BAC)
-Expression vector
Describe one major difference in the organization or content of prokaryotic and eukaryotic genomes.
Prokaryotes with larger genomes will have more genes. In eukaryotes, there is little association between genome size and number of genes.
The full-length (i.e., containing the entire protein coding region) cDNA for a specific eukaryotic gene in humans is 1500 nucleotides long. You screen a pig genomic library with this cDNA and isolate two genomic clones of different lengths. Both clones are sequenced and found to be 1900 and 2100 nucleotides long from start codon to stop codon. How would you explain the presence of two genomic clones in pigs, and the discrepancies in their length compared to the cDNA probe?