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Module 4 Exam - Sheet 1

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What are three key differences between a genomic and a cDNA library?
A genomic library contains all the DNA sequences found in a genome. A cDNA library contains all the DNA sequences that are transcribed into mRNA. A genomic library must contain a large number of clones to ensure that all DNA sequences are represented. A cDNA library is enriched with fragments from actively transcribed genes and introns do not interrupt the cloned sequences
What are Northern analyses used for? Describe the steps involved in performing a Northern analysis, and describe how levels of gene expression are determined.
Northern analyses are used for screening mRNA to determine expression characteristics of a gene. mRNA is extracted, isolated and then separated by electrophoresis. After electrophoresis, the sample is transfered to a nylon membrane and transfered via capillary action. Levels of gene expression can be determined using microarrays.
What is the purpose of an antibiotic resistance gene in a plasmid cloning vector?
it is a selectable marker to distinguish hosts with or without the vector
What is the purpose of the LacZ gene in a plasmid cloning vector?
it is a commonly used selectable marker used with plasmids. It assists in differentiating bacterial plasmids with the specified target gene from those without it by dividing them into blue and white colonies.
During gel electrophoresis, __ will migrate more rapidly than __.
small DNA fragments, large DNA fragments
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
The smallest number of clones that represents the entirety of the genome are called what?
gene library
All of the following are characteristics of the genomics revolution EXCEPT____________.
inability to understand single genes
Which of the below are not steps in the production of genome sequence maps:
isolate the whole chromosome
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.
What do PCR, reverse transcription, and dideoxy DNA sequencing all have in common?
All produce DNA chains as a product
Which of the following are the important proteins needed for cloning a eukaryotic gene into a bacterial plasmid?
B)restriction enzymes specific for the target genesC)DNA ligase
List two especially useful characteristics of cloning vectors.
high copy number, antibiotic resistance genes
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
What is bioinformatics?
a method that uses very large national and international databases to access and work with sequence information
A typical prokaryotic genome has
1 million base pairs of DNA, containing 1000 genes.
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
A set of overlapping DNA fragments that form a contiguous stretch of DNA is called a _________.
contig
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
Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky?
single-stranded complementary tails
What is the enzymatic function of restriction enzymes?
to cleave nucleic acids at specific sites
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?
polylinker
If a restriction enzyme cuts a circular DNA into three fragments, how many restriction sites are there in the DNA?
three
PCR is:
a technique for amplifying DNA sequences in vitro
Of the DNA sequences below, which would probably be the harder to determine?
CGATATATATATATATACGAT. (The repetitive region would make it harder to determine with certainty, even though it is shorter than the other sequence.
Compare the transcriptome of an organism with the proteome. What is described by each?
The transcriptome of an organism is a set of every RNA produced in that organism. The Proteome is the set of every protein produced in that organism.
Rank from "roughest" → "fine detail" the amount of resolution allowed by the following methods of mapping:
__1__Sequence map__4__Cytogenetic map__2__Restriction map__3__Linkage map4 is roughest. CLRS
Match each term with the best letter choice:real-time PCR
DNA quantification
What methods are used to produce mutations in a forward genetics approach?
UV light, EMS and nitrosoguanine
transgene
J. foreign DNA
Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot one generally
hybridizes filter-bound DNA with a DNA probe
Before sequencing, the DNA fragment was cloned into a plasmid. On the strand that acted as the template in the sequencing reaction, what base of the cloned fragment was closest to the primer?
A
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
A BLAST search is done to
find similar gene or protein sequences.
The set of all proteins encoded by the genome is called the _______ .
proteome
For a physical map of a chromosome, distances are measured in units of
base pairs
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, eliminates
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
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
Describe the standard PCR method (in sufficient detail) and how this process is able to produce clones in a "cell-free" system.
The target DNA is denatured using heat, giving two single strands. Primers are added to flank the DNA of interest, and extended using Taq DNA polymerase. The result is two new pieces of whole DNA containing the targeted gene. This is repeated 25-40 times to yield the desired amount of clones. Because all of these cellular machinery can run in this artificial environment, it does not require a host cell to proceed.
cloning vector
plasmid
This is the study of "genes in their entirety."
genomics
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
Another word for a "DNA chip" (microscopic spots of oligonucleotides bound to glass that can be fluorescently labelled to identify levels of expression).
DNA microarray
shuttle vector
multiple hosts
Compare the fields of structural, functional, and comparative genomics. What is the purpose of each?
structural - 3D structure of all proteins in a genomefunctional - interactions of DNA, proteins, and transcriptional factorscomparative - functional genomics between species
A map of the order, overlap and orientation of physically isolated pieces of the genome.
physical map
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
Which of the following enzymes is used to make complementary DNA (cDNA) from RNA?
reverse transcriptase
Plasmids are important in biotechnology because they are
a vehicle for the insertion of foreign DNA into bacteria
Explain why genetic and physical map distances may differ in relative distances between two genes on a chromosome.
A genetic map uses linkage to give the distances between genes. These distances are based on recombination frequency, thus they are not exact physical figures. A physical map is the precise physical location of genes on a chromosome and is not based on recombination.
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?
29
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?
These 4 base single stranded overhangs must be complimentary for them to anneal and be sealed by DNA ligase
ß-galactosidase
lac-Z
A gene construct that indicates when transcription occurs because the protein is easily identified (often GUS or GFP).
reporter gene
YAC
centromere
List four uses of PCR
Idenification of RE variants, Microsatalite analysis, screening for genetic disorders, forensics, paleobiology
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?
Creating a cDNA library of each organism would allow this. This can be done by using reverse transcriptase to create dna transcripts of mRNA with small hairpin loops at the end. DNA polyermase will then synthesize a complement strand. This method gives a library of all expressed genes
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?
Creating a cDNA library of each organism would allow this. This can be done by using reverse transcriptase to create dna transcripts of mRNA with small hairpin loops at the end. DNA polyermase will then synthesize a complement strand. This method gives a library of all expressed genes
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 a unit of recombinant frequency. These are different because the different organisms have different rates of crossover between their chromosomes.
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
PstI would result in an average of 4.8 DNA fragments, RsaI would result in an average of 76.9 DNA fragments.An 8-base cutter would cleave every 8 65,536 bases, on average.
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.
The technique used to clone Snuppy involved removing the nuclei and barr bodies from mature oocytes taken from mixed breed female dogs and fusing these oocyctes with donor nuclei from Afghan hound somatic cells. These were then implanted into surrogate mothers, one of which gave birth to Snuppy.Snuppy was confirmed as a clone via microsatellite analysis using 8 dog microsatellites.
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?
This gene was most likely present in a common ancestor, the presence of two copies could be from gene duplication and the increase in length could be due to elongation of repeats included in the sequence.
ParalogHomologOrthologSynteny
Paralog: (3) Genes related by gene duplication in the genome.Homolog: (1) Closely related genes based on sequence and function.Ortholog: (2) Homologus genes of the same locus inherited from a common ancestor.Synteny: (4) Conservation of the same groups of genes in the chromosomes of 2 or more species.
What is a transgenic organism?
An organism that stably carries a foreign gene within its genome
PCR
Taq polymerase
Write the letter of all of the following statements that are NOT true.a.Coding sequences for gene products can be isolated from cDNA libraries.b.Antibodies are used for Northern blot analysis.c.VNTRs are highly conserved in human populations.d.PCR amplification generates large numbers of linear DNA fragments.e.RNA molecules can be used as hybridization probes in Southern blot analysis.
B
Two genes that evolved from the same common ancestral gene, but are now found as homologs in different organisms are called _______________ .
orthologs
A _______________ family is a group of evolutionarily related genes that arose through repeated evolution of an ancestral gene.
gene
A gene construct that indicates when transcription occurs because the protein is easily identified (often GUS or GFP).
reporter gene