Genetics Week 10

Which of the following features on an engineered Ti plasmid ensures the production of a significant amount of mRNA from the introduced gene?

-A highly efficient promoter such as the CaMV promoter -In order to achieve transcription of the introduced gene, it must be associated with a promoter. A highly efficient promoter such as CaMV serves to maximize expression levels.

What naturally occurring feature of the Ti plasmid makes it useful in genetic engineering of plants?

-It is capable of introducing exogenous genes into plant genomes. -T-DNA genes are removed from the Ti plasmid and are replaced with the gene of interest.

Which of the following conditions might suggest that a genomic DNA sequence contains a protein-coding gene?

-Presence of upstream regulatory elements, such as promoters. -Presence of downstream regulatory elements, such as translation termination sites. -Presence of open reading frame (ORF). -Conservation of similar sequences in other species.

In the context of recombinant DNA technology, of what use is a probe?

-Probes are used to identify and/or locate a particular nucleic acid sequence among a pool of sequences. -Probes are used to identify a complementary nucleic acid sequence.

Which of the following statements about proteomics are true?

-There are many more types of proteins than genes. -Number of protein-coding genes is estimated by genome annotation. -Number of produced proteins can be estimated by electrophoresis, mass-spectrometry, and other techniques.Part complete

The role of the primers in PCR is _______.

-To define the target region and provide a 3' end that can be extended by taq polymerase. -Primers bind to end of the target DNA strands, then taq polymerase synthesizes a new strand using the target DNA as a template.

X-Gal is included in the growth medium on which cells transformed with bacterial plasmids are grown. The reason X-Gal is included is to _______.

-identify bacteria that contain a recombinant plasmid -Colonies produced from cells containing a recombinant plasmid are white, whereas colonies from cells containing a nonrecombinant plasmid are blue.

Identify the steps involved in the microarray procedure.

1. Isolate mRNA from both the normal cells and the cancer cells. 2. Use the enzyme reverse transcriptase and fluorescently labeled nucleotides to make cDNA from the starting material isolated in Step 1. 3. Denature the fluorescently labeled molecule created in Step 2, and incubate it with the microarray. The fluorescently labeled molecules from the two different cell types will hybridize to single-stranded DNA on the microarray. 4. Wash and then scan the microarray to measure the fluorescence at each spot on the array.

Order these steps involved in sequencing using a map-based cloning approach.

1. digest chromosomal DNA with restriction enzymes 2. clone DNA into various large vectors to create overlapping contigs 3. systematically sequence DNA 4. align sequences to assemble chromosomes

Order these steps involved in whole-genome shotgun sequencing.

1. randomly cut the genome into numerous smaller segments 2. sequence DNA 3. align sequences to form contigs

Which of the following DNA sequences is one strand of a restriction enzyme recognition sequence?

5' GGATCC 3' Correct. The 5' → 3' sequence of the complementary strand would be the same as the 5' → 3' sequence of this strand; i.e., this sequence is symmetrical about the midpoint.

Which one of the following are not hallmarks used to identify genes in an unknown prokaryotic sequence?

5' and 3' intron splice sites

Taq polymerase

A DNA synthesis enzyme that can withstand the high temperatures of PCR

TI plasmid

A Ti or tumour inducing plasmid is a plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce their genetic material to plants.

Genomic library

A collection of clones that contains all the DNA sequences of an organism's genome.

Contigs

A continuous DNA sequence reconstructed from overlapping DNA sequences derived by cloning or sequence analysis.

Which one of the following statements best explains this apparent contradiction?

A few repetitive regions of the genome tend to have highly variable repeat lengths that are used in DNA fingerprinting. The majority of the genome has no sequence changes or only single-nucleotide polymorphisms.

Bioinformatics

A field that focuses on the design and use of software and computational methods for the storage, analysis, and management of biological information such as nucleotide or amino acid sequences.

Knockout (KO) organism

A gene in an organism that is inactivated for the purpose of studying gene function. Sometimes called gene targeting.

Polymerase chain reaction (PCR)

A method for amplifying DNA segments that depends on repeated cycles of denaturation, primer annealing, and DNA polymerase-directed DNA synthesis.

What is a probe in molecular biology?

A probe is a labeled or tagged single strand of DNA or RNA used to locate its complementary sequence. They are commonly used in Southern and Northern blots, as well as in library screening.

Molecular cloning

A set of techniques used to insert recombinant DNA from a prokaryotic or eukaryotic source into a replicating vehicle such as plasmids or viral vectors. Cloning refers to making numerous copies of a DNA fragment of interest, such as a gene.

Cloning Vector

A small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

BLAST (Basic Local Alignment Search Tool)

A software application for comparing sequence data (DNA, RNA, protein) to search for sequence similarities.

Genomics

A subdiscipline of the field of genetics generated by the union of classical and molecular biology with the goal of sequencing and understanding genes, gene interaction, genetic elements, as well as the structure and evolution of genomes.

Single nucelotide polymorphisms (SNPs)

A variation in a single nucleotide pair in DNA, usually detected during genomic analysis. Present in at least 1 percent of a population, a SNP is useful as a genetic marker.

Quantitative real-time PCR (qPCR)

A variation of PCR (polymerase chain reaction) that uses fluorescent probes to quantitate the amount of DNA or RNA product present after each round of amplification.

The human genome contains approximately 20,000 protein-coding genes, yet it has the capacity to produce several hundred thousand gene products. What can account for the vast difference in gene number and product number?

Alternative splicing occurs.

Protein domain (motif)

Amino acid sequences with specific conformations and functions that are structurally and functionally distinct from other regions on the same protein.

Human Genome Project

An international effort to determine the sequence of the human genome, to identify all genes in the genome, and to map all genes to specific chromosomes, among other goals.

Transgenic (knock-in) organism

An organism whose genome has been modified by the introduction of external DNA sequences into the germ line (sometimes called knock-in organism).

Annotation

Analysis of genomic nucleotide sequence data to identify the protein-coding genes, the nonprotein-coding genes, and the regulatory sequences and function(s) of each gene.

Transcriptome analysis

Analyze the expression patterns of all genes simultaneously under various growth conditions, times during development, when a regulatory protein is mutated, etc.

Orthologous genes (orthologs)

Any of two or more homologous gene sequences found in different species related by linear descent.

What advantages do BACs and YACs provide over plasmids as cloning vectors?

BACs and YACs: -DNA inserts from 100 kb to 1000 kb can be cloned Plasmids: -easy to separate from the host bacterial chromosome -can only accept DNA pieces up to 25 kb -can use only bacteria as hosts

What is BLAST used for?

BLAST is used to search through databases of DNA and protein sequences to identify portions that align with or are the same as existing sequences.

What is bioinformatics?

Bioinformatics is an interdisciplinary field that develops and improves upon methods for storing, retrieving, organizing and analyzing biological data.

Copy-number variation (CNV)

DNA segments larger than 1 kb that are repeated a variable number of times in the genome.

During a PCR, heat is provided to inactivate the polymerase enzyme.

False

Humans have more DNA and more genes than any other organism.

False

In general, the main goal of cloning is to include as many different genes as possible in a single cloning vector.

False

Paralogous genes (paralogs)

Genes whose origin lies in a gene duplication event within an extant or ancestral species

Yeast artifical chromosome (YAC)

Genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid.

Compare and contrast genomic and cDNA libraries.

Genomic libraries only: -contain noncoding DNA -likely to contain the entire genome cDNA libraries only: -originate from mRNA -do not contain introns -dependent on expression levels of genes -dependent on tissue type

In which case are contigs suspected to be part of the same chromosome?

If their end sequences overlap.

Which factors make yeast artificial chromosomes (YACs) an excellent cloning tool?

In their linear form, YACs contain telomeres at each end for stability, an origin of replication, a yeast centromere, and they can be used to clone up to 1000 kb of DNA.

2-D polyacrylamide gel electrophoresis (2D- PAGE)

Method for separating polypeptides in two dimensions, first by size (molecular weight) and second by electrical charge (isoelectric point).

Plasmid vector

Modified small circular DNA molecule that replicated independently of the genome that are used extensively as vectors for DNA cloning and the production of transgenic plants.

Which one of the following are not hallmarks used to identify genes in an unknown eukaryotic sequence?

Operator sequence

The human insulin gene contains a number of sequences that are removed in the processing of the primary RNA transcript. In spite of the fact that bacterial cells cannot excise these sequences from primary RNA transcripts, explain how a gene like this can be cloned into a bacterial cell and produce insulin.

Plasmids containing insulin genes derived from cDNA, rather than genomic DNA, would be void of introns. Therefore, intron processing would not be necessary.

1. The beta-globin gene has sequence similarity to the other genes in the beta globin family, but it lacks a start codon

Pseudogene

The olfactory receptor gene family in humans contains 16 genes. Six of these genes contain mutations such that the genes do not encode functioning proteins

Pseudogene

Structural genomics

Seeks to describe the 3-dimensional structure of every protein encoded by a given genome. This genome-based approach allows for a high-throughput method of structure determination by a combination of experimental and modeling approaches.

Phage vector

The bacteriophage transferring the genetic material between bacteria

Why is this discipline essential for studying genomes?

The efficiency of genome studying depends on the ability to store, efficiently share, and obtain the maximum amount of information from protein and DNA sequences.

Whole-genome (shot-gun) sequencing

The entire genome is chopped into fragments using restriction enzymes, the fragments are cloned and sequenced. Computers analyze the fragments to put the sequence together.

Proteomics

The entire set of proteins expressed by a cell, tissue, or organism at a given time. The study of the proteome is referred to as proteomics.

Blue-white colony screening

The identification of a colony containing a desired gene by inserting the desired gene into the LacZ gene and plating the colonies on media with the X-gal substrate to differentiate blue colonies that lack the gene from white colonies that contain the gene

Genome

The set of hereditary information encoded in the DNA of an organism, including both the protein-coding and non-protein-coding sequences.

Metagenomics

The study of DNA recovered from organisms collected from the environment as opposed to those grown as laboratory cultures. Often used for estimating the diversity of organisms in an environmental sample.

A _______ organism can be created to express an inserted gene of interest, which may come from a different species.

Transgenic

A common term for a plasmid or other DNA element that serves as a cloning vehicle is vector.

True

In a PCR, primers are complementary to stretches of DNA with which they anneal.

True

One of the primary reasons for generating a large number of different clones in a eukaryotic genomic library is that each vector can take up only a relatively small fraction of the eukaryotic DNA.

True

The products of restriction digestion can be visualized by gel electrophoresis, which separates fragments based on their size.

True Restriction digestion produces fragments of DNA, and the sizes of these fragments can be determined by gel electrophoresis using standard DNA fragments of known size.

Two-dimensional gel electrophoresis is used in proteomics to __________.

Two-dimensional gel electrophoresis is used to separate proteins, first based on charge and then based on their molecular weight.

Which of the following statements is not true? -Alignment can be difficult in repetitive regions of the genome. -Whole-genome sequencing relies on the generation of non-overlapping fragments that are sequenced and aligned to form contigs. -Compared to whole-genome sequencing, the map-based approach is somewhat cumbersome and time-consuming. -Whole-genome sequencing has become the most common method for assembling genomes, with map-based cloning being used to resolve the problems often encountered during whole-genome sequencing.

Whole-genome sequencing relies on the generation of non-overlapping fragments that are sequenced and aligned to form contigs.

Bacterial artificial chromosome (BAC)

a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division

Microarray analysis

a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes.

cDNA library

a combination of cloned cDNA fragments inserted into a collection of host cells, of which constitute some portion of the transcriptome of the organism and are stored as a "library".

Comparative genomics

a field of biological research in which the genomic features of different organisms are compared. The genomic features may include the DNA sequence, genes, gene order, regulatory sequences, and other genomic structural landmarks.

Functional genomics

a field of molecular biology that attempts to make use of the vast data generated by genomic and transcriptomic projects (such as genome sequencing projects and RNA sequencing) to describe gene (and protein) functions and interactions.

Reverse transcription PCR (RT-qPCR)

a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction. It is primarily used to measure the amount of a specific RNA

Epigenome

a multitude of chemical compounds that can tell the genome what to do. The human genome is the complete assembly of DNA (deoxyribonucleic acid)-about 3 billion base pairs - that makes each individual unique.

Multiple cloning site

a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid.

Mass spectrometry

an analytical technique that measures the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio.

What is the definition of a clone?

an identical organism, cell, or molecule that has been derived from a single ancestor

In the context of molecular genetics, reverse transcription PCR (RT-PCR) refers to ________.

assembling a DNA sequence from an RNA template

How do high-throughput techniques such as computer-automated next-generation sequencing and mass spectrometry facilitate research in genomics and proteomics?

by allowing rapid analyses and deployment of genomic information

Which of the following genetic engineering techniques involves restriction enzymes?

cloning DNA into vectors Restriction enzymes are used to cut both the vector DNA and the DNA fragment to give them compatible ends for ligation.

pBluescript II

cloning vectors designed to simplify commonly used cloning and sequencing procedures, including the construction of nested deletions for DNA sequencing, generation of RNA transcripts in vitro and site-specific mutagenesis and gene mapping.

The Human Genome Project, which got underway in 1990, is an international effort to ________.

construct a physical map of the billions of base pairs in the human genome

Homologous genes

genes derived from the same ancestral gene that have accumulated random mutations that make their sequences slightly different

Contig alignment

he process of assembling many sequence fragments into one long genomic sequence or a few long contigs

What is one especially useful characteristic of cloning vectors?

high copy number and antibiotic resistance gene(s)

A ________ organism can be created to terminate the expression of a gene. It can be achieved via ______ by swapping the gene of interest with a copy that will produce a nonfunctional protein.

knockout, recombination

Compared with prokaryotic chromosomes, eukaryotic chromosomes are ________

large, linear, less densely packed with protein-coding genes, mainly organized in single gene units with introns

A duplicated gene in mosquitos accumulated mutations that allow it to break down a chemical found in a common pesticide

neofunctionalization

Proteomics is the ________.

process of defining the complete set of proteins encoded by a genome

How do results from the PGP differ from those of the HGP?

results from the PGP: -provides sequence information on individuals -provides individual sequences of diploid genomes results from the HGP -involves a haploid reference genome, thus underestimates the extent of genome variation between individuals

Hemoglobin consists of an alpha chain and a beta chain, each produced by a different but similar gene. Neither gene alone can make a functional hemoglobin molecule

subfunctionalization

In most vertebrates, the pax transcription factor acts during development in the eye and brain. In zebra fish, each tissue has a different but similar transcription factor (paxA and paxB) and both transcription factors are necessary for normal development

subfunctionalization

Library screening

the process of identification of the clones carrying the gene of interest. Screening relies on a unique property of a clone in a library. The DNA libraries consist of a collection of probably many thousand clones in the form of either plaques or colonies on a plate

Which of the following is NOT a reason for generating transgenic crops? -to provide crop resistance against insect and viral pests, drought, and herbicides -to improve the growth characteristics and yield of agriculturally valuable crops -to increase the number of different plant species used in agriculture -to increase the nutritional value of crops

to increase the number of different plant species used in agriculture