Mol Gen Mod 4
Terms in this set (76)
bacterial artificial chromosome or BAC,
List at least three different kinds of bacterial cloning vectors.2-2Plasmid
Cell free cloning,
Identification of restriction enzyme variants,
Screening for genetic disorders
Diagnostic screening for infectious organisms,
List four uses of PCR 10-3
Origin of replication for propagation in the host
, Selectable marker like Amp resistance, Polylinker or unique restriction enzyme sites to facilitate cloning
List the three basic components required for a bacterial cloning vector and briefly describe the purpose of each.1-2
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? 9-2
There was likely a duplication of this gene in pigs,
Also duplication the gene was diverged,
Or humans lost one copy of the 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?8-3
Transcriptome, Proteome, 3
The transcriptome of a genome contains more components than the proteome. Explain why this is true.
_______________: all the RNA molecules transcribed from a genome
__________: all the proteins encoded by the genome
It takes _ RNA codons to code for one protein, so it is natural to have more transcriptome (RNA molecules) than proteome (proteins).
What are Northern analyses used for? Describe the steps involved in performing a Northern analysis, and describe how levels of gene expression are determined:
(Know step by step)
detection and quantitation of _____ levels
____ isolation (total or poly(A) RNA)
Denaturing agarose gel electrophoresis
Transfer to solid support and immobilization
Prehybridization and hybridization with probe
Stripping and reprobing (optional)
cDNA library, genomic, CDNA, cDNA, mRNA
What are three key differences between a genomic and a cDNA library?4-2
________. Represent only transcribed regions of the genome
All genes equally represented in _______ library while ________ library reflects the level of expression of a gene in a particular cell type or tissue
________ library contained only sequences found in the mature ______- introns are removed
UV light, EMS, Nitrosoguandandine, transposons
What Method are used to produce mutations in a forward genetics approach?
They consist of highly repetitive DNA, and so strand slippage issues can confuse the determination of a consensus sequence.
Why are telomeres and centromeres particularly difficult to sequence?9-3
28 (29 or 30)
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? 5-2
The RE must have complementary sticky ends so that they will anneal together
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?1-14
A _______________ family is a group of evolutionarily related genes that arose through repeated evolution of an ancestral gene. 3-8
A gene construct that indicates when transcription occurs because the protein is easily identified (often GUS or GFP).2-8
A map of the distribution of cloned genomic DNA from genomic clone libraries.3-10
A map of the order, overlap and orientation of physically isolated pieces of the genome.3-5
Another word for a "DNA chip" (microscopic spots of oligonucleotides bound to glass that can be fluorescently labelled to identify levels of expression).1-4
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.3-7
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?
Gene mapped based on recombination will appear to be very close together in ____________ regions due to low rates of recombination.
Distances between the same genes on the physical map may be much ________ when compared to their regions of the chromosomes
Immunoglobin gene subunit diversity
,Differential splicing to create diversity
,Break and nibble mechanism to increase diversity
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.
You could generate cDNA libraries and compare the transcribed regions of the genome
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? 3-3
Minimum tiling path
The smallest number of clones that represents the entirety of the genome are called what? 2-13
This is the study of "genes in their entirety."
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.
Two genes that evolved from the same common ancestral gene, but are now found as homologs in different organisms are called _______________ .
a transgenic organism
An organism that stably carries a foreign gene within is genome
To determine if the vector is present in host cell
What is the purpose of an antibiotic resistance gene in a plasmid cloning vector?
To be able to distinguish those colonies with an insert using blue/white selection
white have an insert
What is the purpose of the LacZ gene in a plasmid cloning vector?
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?2-17
5' TGCTAGC 3'
5' CGATCGT 3'
5' GCTAGCA 3'
5' ACGATCG 3'
For a physical map of a chromosome, distances are measured in units of
In the previous list of cloned fragments, the fragments needed to make the longest possible contig, with the least amount of overlap, are .1-9
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
Match each term with the best letter choice: ß-galactosidase
In situ hybridization
a, it's more repetitive
Of the DNA sequences below, which would probably be the harder to determine?1-26
A linear DNA fragment is cut with a restriction enzyme to yield two fragments. There is/are __ site(s) for this enzyme in this fragment.
paralog, otholog, synteny, homolog
1closely related gene based on sequence and function
2 homologous gene of the same locus inherited from a common ancestor
3. gene related by gene duplication in the genome
4.conservation of the same group of genes in the chromosomes of 2 or more species
Rank from "roughest" → "fine detail" the amount of resolution allowed by the following methods of mapping:1-7
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?1-17
Eukaryotic: repetitive DNA
Eukaryotic: circular DNA
Prokaryotic: presence of plasmids
Prokaryotic: repetitive DNA
A BLAST search is done to2-26
find the chromosomal location of a sequence.
predict the three-dimensional structure of a protein from its amino acid sequence.
find similar gene or protein sequences.
find restriction sites and SNPs in a sequence.
determine the conditions under which a gene is expressed.
A human gene with a disease phenotype is going to be mapped by positional cloning. Which would be the most useful for this task?1-33
An EST database of the human genome
Microarray data of tissues in which the gene is expressed
Information about bacterial orthologs of the gene
Data about the inheritance of SNP markers in families with the disease
Whole-genome-shotgun clones of the human genome
All of the following are characteristics of the genomics revolution EXCEPT_____________2-23
Enabled reverse genetics approach to genetics research
Facilitated collaborative research networks
Ability to conduct discovery-based research
Large scale acquisition of DNA sequences
Inability to understand single genes
A principal problem with inserting an unmodified mammalian gene into a bacterial plasmid, and then getting that gene expressed in bacteria, is that 1-27
bacterial DNA is not found in a membrane-bounded nucleus and is therefore incompatible with mammalian DNA
bacterial RNA polymerase cannot make RNA complementary to mammalian DNA
prokaryotes use a different genetic code from that of eukaryotes
bacteria cannot remove eukaryotic introns.
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?1-35
A 10-kb, an 8-kb, and a 2-kb fragment
A 10-kb and a 2-kb fragment
A 12-kb fragment
An 8-kb and a 2-kb fragment
An 8-kb fragment
A set of overlapping DNA fragments that form a contiguous stretch of DNA is called a _________.2-21
A typical prokaryotic genome has2-20
1000 base pairs of DNA, containing a few thousand genes.
1 million base pairs of DNA, containing a few hundred genes.
1000 base pairs of DNA, containing a few hundred genes.
1 million base pairs of DNA, containing 1000 genes.
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?1-29
During gel electrophoresis, __ will migrate more rapidly than __. 2-10
a. cloning vectors
b. ethidium bromide
c. large DNA fragments
d. DNA size markers
e. small DNA fragments
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 by1-34
Identifying the molecular weights of the fragments in question
Measuring the sizes of the bands on the gel
None of the above
Removing the bands from the gel and hybridizing them with a known strand of DNA complementary to the gene of interest
Knowing the isoelectric points of the piece in question.
If a restriction enzyme cuts a circular DNA into three fragments, how many restriction sites are there in the DNA?1-32
List two especially useful characteristics of cloning vectors. 2-24
ability to integrate into the host chromosome and then cause a lytic cycle
nonautonomous replication and transposition
high copy number and antibiotic resistance gene(s)
reverse transcriptase and ligase activities
virulence and lysogenicity
Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot one generally 1-28
hybridizes filter-bound DNA with a DNA probe
hybridizes filter-bound RNA with a DNA probe.
examines amino acid substitutions with radioactive probes.
cleaves RNA with restriction endonucleases
ligates DNA with DNA ligase.
One of the primary reasons for the necessity of generating a large number of clones in a eukaryotic genomic library is that 2-16
each ligation product is sequence specific.
the host range of the vector is limited
each cosmid replicates nonautomously.
each vector can take up only a relatively small fraction of the eukaryotic DNA.
lysogenic phage continue to integrate their DNA into the host chromosome, thus reducing the number of desired recombinant clones.
one of the control elements of the cell cycle
None of the above
necessary for efficient replication of cell's DNA in interphase
a technique for amplifying DNA sequences in vitro
Plasmids are important in biotechnology because they are2-25
recognition sites on recombinant DNA strands
surfaces for respiratory processes in bacteria
surfaces for protein synthesis in eukaryotic recombinants
a vehicle for the insertion of foreign genes into bacteria
Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky? 1-22
single stranded complementary tails
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? 1-25
The set of all proteins encoded by the genome is called the _______ .1-21
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.1-31
visual examination, eliminate
temperature extremes, enhance
epistasis analysis, enhance
complementation analysis, enhance
chemical removal, activate
What do PCR, reverse transcription, and dideoxy DNA sequencing all have in common? 1-19
All produce lipid as a product.
All produce RNA as a product.
All produce DNA chains as a product.
All produce RNA as a product.
What is bioinformatics?1-30
a technique using 3D images of genes in order to predict how and when they will be expressed
a method that uses very large national and international databases to access and work with sequence information
a series of search programs that allow a student to identify who in the world is trying to sequence a given species
a software program available from NIH to design genes
What is the enzymatic function of restriction enzymes? 2-22
to cleave nucleic acids at specific sites
to add new nucleotides to the growing strand of DNA
to join nucleotides during transcription.
to repair breaks in sugar-phosphate backbones
What is the function of dideoxynucleotides in Sanger DNA sequencing?1-20
They stop synthesis at a specific site, so the base at that site can be determined.
They cut the sequenced DNA at specific sites.
They allow only the specific sequencing of the RNAs of a genome.
They act as primers for reverse transcriptase.
They act as primers for DNA polymerase.
Which of the following are the important proteins needed for cloning a eukaryotic gene into a bacterial plasmid?1-23
restriction enzymes specific for the target genes
Both B and C
Which of the following enzymes is used to make complementary DNA (cDNA) from RNA? 1-
isolation of stem cells from a lamb embryo and production of a zygotic equivalent
Which of the below are not steps in the production of genome sequence maps:2-18
Identify molecular markers on specific chromosomes.
Isolate whole chromosomes.
When sequences are obtained, assemble and organize the sequences in order.
All of these are steps you would use.
Read the sequence of individual piece of the genome.
Which technique would NOT be used to find a gene for a functional protein in a sequenced region of a genome?1-18
See if a SNP database contains sequences in the region.
Scan the region for intron splice sites.
Scan the region for ORFs.
Scan the region for promoter
See if an EST database contains sequences in the region.
Write the letter all of the following statements that are NOT true.1-6
Coding sequences for gene products can be isolated from cDNA libraries.
Antibodies are used for Northern blot analysis.
VNTRs are highly conserved in human populations.
PCR amplification generates large numbers of linear DNA fragments.
RNA molecules can be used as hybridization probes in Southern blot analysis.
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.
1400bp/E1&E2 1600 bp
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