Module 3 Review and Practice Exam Flashcards
You determine that you have only 3 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?
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29
What enzyme is required for PCR? State the specific name of the enzyme, not just the type of enzyme.
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Taq polymerase
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.
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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.
Single stranded template DNA; two specific primer molecules; the two primer molecules have to be complementary to part of the template DNA molecule.
Name the 3 basic steps of PCR and describe the molecular processes that occur in each. How is each step induced?
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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.
- Denature target DNA, forming a single strand template DNA molecule. This process is induced by raising the temperature to separate two DNA strands.
- Anneal specific primers to flank DNA target of interest. These primers are complementary to the template DNA. This process is induced by cooling down the environmental temperature and let the primer molecule form hydrogen bonds with the template DNA molecule.
- Extension of primer by Taq DNA polymerase, creating a complementary strand of the template DNA molecule.
How does this system work to amplify DNA?
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Each run of PCR results in double the amount of initial DNA, thus after several runs, the initial target DNA is significantly amplified.
The system works to amplify DNA, by the addition of enzymes to the targeted region which allow it to be flanked, then synthesized by the Taq polymerase. After each trial the taregt region is synthesized at an exponential rate.
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.
This answer it true (feedback)
Two genes that evolved from the same common ancestral gene, but are now found as homologs in different organisms are called ____________ .
orthologs
PCR
Taq polymerase
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.
genome project
During gel electrophoresis, __ will migrate more rapidly than __.
During gel electrophoresis, small DNA fragments will migrate more rapidly than large DNA fragments.
In the previous list of cloned fragments, the fragments needed to make the longest possible contig, with the least amount of overlap, are…
Cloned fragment, Markers present A, M4 M5 B, M1 M2 M3 C, M6 M7 M8 D, M3 M4 M5 M6 E, M3 M4 M5 F, M8 M9 M10
b,d,c,f
B, M1 M2 M3
D, M3 M4 M5 M6
C, M6 M7 M8
F, M8 M9 M10
Which of the following enzymes is used to make complementary DNA (cDNA) from RNA?
reverse transcriptase
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.
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.
Which techniques would be used to find a gene for a functional protein in a sequenced region of a genome?
Scan the region for promoter sequences.
Scan the region for ORFs.
Scan the region for intron splice sites.
See if an EST database contains sequences in the region.
List two especially useful characteristics of cloning vectors.
high copy number and antibiotic resistance gene(s)
0.1% frequency of recombination is observed ….
….in genes located very close to one another on the same chromosome.
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
The set of all proteins encoded by the genome is called the _______ .
proteome
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.
The difference between a genetic screening experiment and a selection experiment is that a screening experiment involves visual examination, whereas a selection experiment creates conditions that eliminate irrelevant organisms.
You are handling a paternity lawsuit brought against five potential fathers by a woman. You isolated DNA from the mother, the child, and all the potential fathers. After using PCR to amplify specific polymorphic loci from each individual, you fractionate the amplified products on an agarose gel and stain with ethidium bromide to visualize the DNA fingerprints (shown below). Mo = mother; Ch = child; M1–M5 = potential fathers. Do these results confirm that any of the men are the child’s biological father? Explain your answer.
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Any band that the child has that the mother has as well is considered uninformative. (this doesn’t mean that that particular one actually came from the mother - it could have come from the father if he has it as well). Any band that the child has that is absent in the mother must have come from its father. Based, on this, M4 is the child’s father.
Compare the fields of structural, functional, and comparative genomics. What is the purpose of each?
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structural genomics is the organization and sequece of geneic info in a genome. functional genomics is tells what the sequences do and compartive genomics compares and contrast differences in gene sequences
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?
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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.
A map unit is the amount of measured recombination b/w two link points. One map unit in a human is many more bp than in yeast due to the repitition in human genes. The amount of homologous pairs in humans must be lower than yeast.
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.
Primer walking
real-time PCR
DNA quantification
The smallest number of clones that represents the entirety of the genome are called what?
minimum tiling path
A _______________ family is a group of evolutionarily related genes that arose through repeated evolution of an ancestral gene.
multigene
Which of the below are NOT steps in the production of genome sequence maps:
Isolate whole chromosomes.
A fragment of DNA is cloned into a plasmid with a sequencing primer binding site. After dideoxy sequencing, the gel pattern shown in this diagram is obtained. What was the sequence of the DNA strand that acted as the template in the sequencing reaction?
5’ GCTAGCA 3’
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 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
If a restriction enzyme cuts a circular DNA into 3 fragments, how many restriction sites are there in the DNA?
3
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
For a physical map of a chromosome, distances are measured in units of
base pairs.
Which are steps in the production of genome sequence maps:
When sequences are obtained, assemble and organize the sequences in order.
Identify molecular markers on specific chromosomes.
Read the sequence of individual piece of the genome.
2 of the 7 different pea traits examined by Mendel involved genes that we now know are linked. Knowing this, can you explain why Mendel was still able to use results from his crossing experiments to develop the principle of independent assortment?
The genes are linked on the same chromosome, but are far enough apart ( > 50 map units) from each other so that they still sort independently.
2 of the 7 different pea traits examined by Mendel involved genes that we now know are linked. Knowing this, can you explain why Mendel was still able to use results from his crossing experiments to develop the principle of independent assortment?
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The genes are linked on the same chromosome, but are far enough apart ( > 50 map units) from each other so that they still sort independently.
Compare the transcriptome of an organism with the proteome. What is described by each? Which one will generally have more macromolecules, and why?
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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.
Transcriptome - set of all RNA transcripts produced by the genome at any one time. Bigger.
Proteome - complete set of proteins expressed during a cell’s lifetime.
It takes 3 RNA codons to code for one protein, so it is natural to have more RNA molecules (transcriptome) than proteins (proteome).
The transcriptome is all of the RNA transcribed from the DNA in an organism, the proteome is all of the proteins in an organism. The transcriptome will generally have more macro molecules, because not all of the transcripts produce (e.g. introns) will code for something.
The transcriptome identifies all of the RNA transcripts in a genome. The proteome identifies all of the proteins that are encoded by that genome. The transcriptome will generally have more macromolecules because it also includes all non-coding RNA.
Explain why the greatest diversity of human SNPs is found among African people.
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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.
Humans are thought to have first evolved in Africa. These populations are the oldest with the greatest amount of time to accumulate polymorphisms
The “distance” between two linked gene pairs can be expressed as a percentage. Name the unit based on percent recombination that was created in honor of the scientist who pioneered the use of fruit flies for genetic research.
centimorgan
YAC
centromere
This is the study of “all genes in an organism in their entirety.”
genomics
The following statements are NOT true.
Antibodies are used for Northern blot analysis.
VNTRs are highly conserved in human populations.
The following statements are TRUE.
Coding sequences for gene products can be isolated from cDNA libraries.
PCR amplification generates large numbers of linear DNA fragments.
RNA molecules can be used as hybridization probes in Southern blot analysis.
ß-galactosidase
lacZ
Between which 2 genes would you expect the highest frequency of recombination?
A____W__E_________G
A and G
Plasmids are important in biotechnology because they are
a vehicle for the insertion of foreign genes into bacteria
A human gene with a disease phenotype is going to be mapped by positional cloning.
Which would be the most useful for this task?
Data about the inheritance of SNP markers in families with the disease
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 | 200 | 1400
