Genetics Test 4
Example of a palindromic sequence and how restriction enzymes deal with palindromic sequences
5'-AAGCTT-3' 3'-TTCGAA-5' - Palindromic sequences are 2 different strands, not forward and backward on the same strand **Restriction enzymes recognize palindromic sequences
List 3 techniques that can be used to study the expression of a gene
Microarrays RNA sequencing Reporter sequences
Differentiate between ortholog and paralog
Orthologs - Homologous genes in different species that evolved from a common ancestor Paralogs - homologous genes in the same organism that arose through duplication
Define Probe
Piece of DNA that is complementary to sequence being searched for - has a label on it
Define transformation
Process in which DNA (plasmid) is taken up by a bacterial cell
Define restriction enzyme (restriction endonuclease)
Restriction enzymes are able to make double stranded cuts at specific sequences (called restriction sites) Restriction enzymes are produced naturally by bacteria and are used in defense against viruses
Define metagenomics
Sequence genomes of an entire group of organisms that share a common environment
Differentiate between dNTP and ddNTP
dNTP - missing 1 OH group ddNTP - missing 2 OH groups
Define plasmid
plasmids- small circular DNA molecules found in bacteria *Plasmid vectors are genetically engineered as vectors
List the 5 components of PCR
template DNA dNTPs DNA polymerase Salts/Buffers Primers
Describe how reporter sequences can provide information about the location of gene expression
- Clone gene with its regulatory sequences - Replace coding region with a reporter gene (ie, GFP) - Insert into genome to create transgenic organism - Observe location of the reporter
Describe the process of RNA Seq
- Collect total RNA from cells - Isolate mRNA - Reverse transcribe into cDNA - Fragment cDNA - Sequence with next-gen sequencing - Assemble sequences into RNA transcripts
Explain in situ hybridization
- Fix cell to slide - Denature DNA/RNA - Add probe that fluoresces
Define functional genomics
- Functional genomics characterizes what sequences do their functions - Determining the function of DNA sequences * Identify RNA transcribed from genome (transcriptome) * Identify proteins encoded by genome (proteome) - homology searches can be used to determine function
Define transcriptome and proteome
- Identify RNA transcribed from genome (transcriptome) - Identify proteins encoded by genome (proteome)
Define bioinformatics
- Molecular biology + computer science - Identify genes - Annotate genes
Define PCR
- Polymerase Chain Reaction - Method for amplifying DNA "Replication in a test tube"
PCR Recipe - What doesn't belong - Template DNA - Ribosome - dNTPs - amino acids - DNA polymerase - tRNA - Salts/Buffers - Primers - Restriction enzymes
- Ribosome - Amino acids - tRNA - restriction enzymes
Describe how CRISPR-Cas9 is used for genome editing
- Scientists have engineered crRNA from bacteria with a sequence that is specific to the target sequence of interest - The other part of the crRNA contains a sequence that pairs with CAS9 protein = crRNA/Cas9 complex - This complex finds and binds to the target DNA sequence - Cas9 makes double-stranded cuts within the target DNA sequence - The cell's DNA repair mechanisms kick in to try to repair the cut—2 things can happen 1. Nonhomologous end joining is a repair pathway that repairs chromosome breaks but often causes inserts/deletions within the process—this would inactivate the gene 2. Donor DNA can be inserted into the cell with ends complementary to the break sequence—homologous recombination would insert this sequence to repair the gene
Metagenomics
- Sequence genomes of an entire group of organisms that share a common environment
Explain how SNPs can be used for genome-wide association studies
- Use SNPs across the genome to find genes of interest
Describe how microarrays can be used to examine gene expression
- Used to study expression of numerous genes - One way to study which genes are turned on or off in a cell One approach to transcriptomics uses microarrays, which rely on nucleic acid hybridization. In this process, known DNA fragments are used as probes to find complementary sequences. Numerous known DNA fragments are fixed to a solid support in an orderly pattern, or array, usually as a series
Understand how a foreign DNA fragment can be inserted into a plasmid
- We cut our plasmid and gene of interest with the same restriction enzyme - Ligase to seal and get recombinant plasmid
Describe how transgenic animals could be used for the purpose of reverse genetics and how these animals are created
- With transgenic animals, we are not actually mutating the gene, but we are adding a gene into the organism that would normally not be there, and then observing the phenotype - Take the zygote before the nuclei from the egg and sperm fuse, before they fuse they are called pronuclei. We inject the gene of interest into one of the 2 pronuclei, implant it into a pseudopregnant mouse, and then screen the babies for which mice have the gene in it. Since the rats are hetero, we have to breed until we get a homozygous mouse
Define homology search and explain how this can be used to understand the function of a gene
- computational methods that search for similar, evolutionarily related (homologous) genes
List some goals of the Human Genome Project
- develop sequencing technology - sequence genomes of model organisms - determine the sequence of the human genome (3.2 billion bp) - identify all genes in human genome (~20,000-25,000)
List some problems associated with sequencing of an entire genome
- only small fragments (500-700 bp) can be sequenced at a time - must fragment genome into millions of small overlapping fragments for sequencing - difficulty ordering the sequenced fragments
Know that special vectors are required to express a foreign gene- expression vectors- and they need to have sequences that allow the gene to be transcribed and translated in addition to being replicated
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Chapter 19 Learning Objectives
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Chapter 20 Learning Objectives
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Chapter 21 Learning Objectives
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Chapter 19 Comprehension Questions
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Chapter 20 Comprehension Questions
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Cells 1. With no plasmid 2. With intact plasmid 3. With recombinant plasmid Do they have amp resistance, b-gal, and what color are the colonies
1. With no plasmid - No amp resistance, they would die and not show up on the plate 2. With intact plasmid - Amp, b-gal produced, blue colonies 3. With recombinant plasmid - amp, no b-gal, white colonies
Explain how to use selectable markers to identify recombinant cells
2 selectable markers - ampR - LacZ If the bacterial cell did not take up a plasmid, they will not have ampR and will die forming no colonies. If the bacterial cell took up a plasmid without our gene of interest inserted where the lacZ should be, it will be blue, which we do not want
Give the percent similarity between two humans
99.9%
What is the difference between a genetic map and a physical map? Which generally has higher resolution and accuracy, and why?
A genetic map, also known as a linkage map, provides relative positions of genes or markers based on genetic recombination frequencies. A physical map locates genes or markers based on the physical length (in base pairs, kilobases, or megabases) of DNA sequences. Physical maps have higher resolution and accuracy than genetic maps. Because recombination frequencies vary from one region of the chromosome to another, distances in genetic maps are approximate. Genetic maps also have lower resolution because recombination is difficult to observe between loci that are very close to each other (tightly linked). Physical maps based on DNA sequences or restriction sites can provide accuracy and resolution down to a single base pair of a DNA sequence.
How does a genomic library differ from a cDNA library?
A genomic library is generated by cloning fragments of chromosomal DNA into a cloning vector. Chromosomal DNA is randomly fragmented by shearing or by partial digestion with a restriction enzyme. A cDNA library is made from mRNA sequences. Cellular mRNAs are isolated, and then reverse transcriptase is used to copy the mRNA sequences to cDNA, which are cloned into plasmid or phage vectors. Therefore, a cDNA library only represents the genes expressed in the tissues of origin for the RNA. Non-coding parts of the genes, such as promoters, enhancers, and introns, are not included in the cloned sequences. The frequency of different cDNA sequences depends on how highly expressed a gene is in the tissue of origin.
What is a haplotype? How do different haplotypes arise?
A haplotype is a particular set of neighboring SNPs or other DNA markers observed on a single chromosome or chromosome region. These markers tend to be inherited together as a set because of linkage. New haplotypes arise through mutations in SNPs or other DNA markers. Also, meiotic recombination within the chromosomal region can split the haplotype and create new recombinant haplotypes.
Explain how a reporter sequence can be used to provide information about the expression pattern of a gene.
A reporter sequence is one that encodes an easily observable product. The reporter is fused to the gene of interest in such a way that the gene's regulatory sequences drive expression of the observable product. Typically, a segment of genomic DNA that includes the upstream promoter region and other cis-acting regulatory sequences is ligated to the reporter gene sequence. This construct may then be used to create a transgenic organism expressing the recombinant reporter gene fusion. The reporter may have enzymatic activity (like β-galactosidase) that is detectable with a substrate that forms a colored product, or the reporter itself may be fluorescent (like green fluorescent protein). Alternatively, the reporter product may be detected with a fluorescently tagged antibody. The gene's own regulatory sequences specify the developmental pattern of expression of the reporter as they would the native gene. If the protein-coding region of the gene is also included, in part or in full, the resulting translational fusion product can be used to study the subcellular localization of the protein.
How is CRISPR-Cas used to introduce specific changes into DNA sequences?
A single guide RNA (sgRNA) is engineered that combines a tracrRNA with a crRNA and contains approximately 20 nucleotides complementary to the target DNA sequence. The sgRNA binds to a Cas protein (such as Cas9). The sgRNA:Cas complex then functions as a sequence-specific endonuclease. It finds DNA sequences that are complementary to the sgRNA. If the complementary DNA sequence also has a protospacer-adjacent motif (PAM), then the sgRNA:Cas complex creates double-stranded DNA breaks adjacent to the target sequence. The cell then activates its double-stranded-DNA break repair systems. Nonhomologous end-joining splices the broken ends together in the absence of a homologous template and frequently results in small deletions or duplications that will inactivate the target gene. Alternatively, repair by homologous recombination copies the sequence from a homologous template. The latter can be used to create specific changes in an organism's DNA sequence by providing templates with the desired DNA sequence. CRISPR-Cas systems are more specific than restriction enzymes because they recognize longer nucleotide sequences. They are easily programmed against different target sequences just by changing the sequence of the sgRNA. CRISPR-Cas can work in any cell or organism where the sgRNA and Cas can be introduced.
Describe 2 ways that a gene could be mutated for the purpose of reverse genetics
CRISPR-Cas9 Knockout mice
Explain how CRISPR-Cas9 can be used in gene therapy
CRISPR-Cas9 can try to delete the codon causing mutations using nonhomologous end joining or correct to the wild-type sequence using homologous recombination
Define cloning vector and know the 3 elements of a cloning vector
Cloning vector: carried foreign gene into host cell "vector is the vehicle that drives the gene into the bacterial cell" --> the vector is a plasmid Requirements of a cloning vector 1. Origin of replication - put gene into plasmid, we want it to be copied so we need an origin of replication 2. One or more unique restriction sites - need restriction sites for restriction enzymes to cut and put gene into vector 3. Selectable markers - Selectable markers are going to allow us to identify which bacterial cells have the gene we want in it
Give three important characteristics of cloning vectors.
Cloning vectors should have: (1) An origin of DNA replication so they can be maintained in a cell (2) A gene, such as antibiotic resistance, to identify or select for cells that carry the vector (3) A unique restriction site or series of unique sites into which a foreign DNA fragment may be inserted
Describe the 2 types of cuts restriction enzymes can make
Cohesive/sticky ends/staggered - Single stranded overhangs - these types of cuts are easier to come back together Blunt cuts
How are genes recognized within genomic sequences?
Computer algorithms (and humans) recognize or predict genes within genomic sequences by analysis of the following: (1) Open reading frames (ORFs) that include a start and a stop codon in the same frame (2) 5' and 3' splice-site sequences that mark the beginnings and ends of introns (3) Consensus sequences found in promoters (4) Homology (sequence similarity) to previously characterized genes in the same or other species
What is DNA fingerprinting? What types of sequences are examined in DNA fingerprinting?
DNA fingerprinting is the use of highly variable DNA sequences to identify an individual. This is useful for forensic investigations, specifically to determine whether the suspect could have contributed to the evidentiary DNA obtained from blood or other bodily fluids found at the scene of a crime. Other applications include paternity testing and the identification of bodily remains. Today, most DNA fingerprinting makes use of minisatellites, or short tandem repeats (STRs), which are small repeat sequences of just a few nucleotides that vary in number of copies among individuals. STRs are found at many loci throughout the genome and can be amplified by PCR. The variable number of repeats creates PCR fragments of different sizes. Genotyping at 13 to 20 of these unlinked STR loci can identify one individual among trillions of potential genotypes.
After DNA fragments have been separated by gel electrophoresis, how can they be visualized?
DNA molecules can be visualized by staining with a fluorescent dye, such as ethidium bromide, which intercalates between the stacked bases of the DNA double helix. The dye-DNA complex fluoresces when exposed to an ultraviolet light source. Alternatively, DNA molecules can be visualized by attaching chemical labels to the DNA before it is placed in the gel.When genomic DNA is fragmented and separated by electrophoresis and stained, a continuous smear is observed on the gel because of the presence of thousands of fragments of differing sizes. Usually, researchers are interested in only a few of these fragments. A technique known as Southern blotting is a way to locate the desired fragments in the smear of DNA. This technique utilizes a labeled DNA or RNA molecule (a probe) with a base sequence complementary to the DNA fragments of interest. Following electrophoresis, the DNA fragments are denatured and transferred to a permanent solid medium (such as a nitrocellulose or nylon membrane). The membrane is placed in a hybridization solution containing the labeled probe. The probe binds to (hybridizes with) any DNA fragments on the membrane that bear complementary sequences. The location of the probe on the medium is detectable by its label (a radioactive isotope or other chemical tag).
Describe the dideoxy method of DNA sequencing
Dideoxy sequencing (Sanger sequencing) - requires dNTPs and ddNTPs - For all sequencing reactions, they all start w/a PCR reaction - Then we divide the mixture into 4 tubes adding 1 ddNTP to each of the tubes and take it through the PCR process (denaturation, annealing, elongation) - separate fragments by size through gel electrophoresis, keep them in their lanes so we can read the sequence - new strand made 5'-->3' so the shorter pieces will be at the 5' end, read gel bottom to top **Sequence in the gel is the COMPLIMENT to the original sequence, so to get the original do compliment pairing and in an anti-parallel manner
What is the purpose of the dideoxynucleoside triphosphates in the dideoxy-sequencing reaction?
Dideoxynucleoside triphosphates (ddNTPs) act as a substrate for DNA polymerase but cause termination of DNA synthesis when they are incorporated because they lack the 3′-OH for the addition of the next nucleotide. Mixed with regular dNTPs, fluorescently labeled ddNTPs are incorporated at random into growing DNA strands and generate a series of DNA fragments that have terminated at every nucleotide position along the template DNA molecule being sequenced. These fragments can be separated by gel electrophoresis in an automated sequencer. Because each of the four ddNTPs carries a different fluorescent label, a laser detector scanning near the end of the gel can distinguish which base terminates each fragment. Reading the fragments from shorter to longer, the automated DNA sequencer determines the 5' to 3' sequence of the newly synthesized DNA molecule. The template strand of DNA will have the complementary sequence (going from 3' to 5').
Define gene therapy
Direct transfer of genes into humans to treat disease
Genes that are evolutionary related are called a. orthologs b. paralogs c. homologs d. both a and c e. a,b, and c
E
Differentiate between forward and reverse genetics - List the types of ways we can do reverse genetics
Forward genetics - observe phenotype and determine gene Reverse genetics - Mutate a gene and then observe the phenotype - How to mutate a gene: CRISPR-Cas9 or knockout mice - We could use transgenic animals where we add the gene - We could use RNAi where we turn the gene off without mutation
How does a reverse genetics approach differ from a forward genetics approach?
Forward genetics begins with a mutant phenotype and proceeds toward cloning and characterization of the DNA encoding the gene. Reverse genetics begins with the DNA sequence of a gene, then generates specific mutations within that gene and requires the reinsertion of the modified gene back into cells or the whole organism to characterize the functions of the gene through new mutant phenotypes.
Understand how gel electrophoresis can be used to separate DNA fragments by size
Gel electrophoresis separates DNA fragments by size, the smallest pieces of DNA move faster and will be closer to the bottom of the gel When we apply a current, DNA travels through the gel towards the positive end which is furthest from the wells because DNA has a negative charge
Explain how gel electrophoresis is used to separate DNA fragments of different lengths.
Gel electrophoresis uses an electric field to drive DNA molecules through a gel that acts as a molecular sieve. The gel is a porous matrix of agarose or polyacrylamide. DNA molecules are loaded into a slot or well at one end of the gel. When an electric field is applied, the negatively charged DNA molecules migrate toward the positive electrode. Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and migrate faster than longer DNA molecules, which must wind their way around obstacles and through the pores in the gel matrix. Over time, DNA fragments of different sizes have migrated different distances, with the smallest fragments the greatest distances from where the DNA was loaded into the gel.
Define gene cloning and know the process for cloning a gene in bacteria
Gene cloning - produce identical copies (clones) of a gene - Place gene into a host genome (bacteria) to be replicated
Define gene library and differentiate between a genomic library and a cDNA library
Gene library: set of bacterial colonies containing DNA from a single source Genomic library: library containing entire genome cDNA library: library containing DNA sequences complementary to mRNA (DNA sequences that are expressed) --> mRNA to cDNA is reverse transcription
What is gene therapy?
Gene therapy is the direct transfer of functional genes into somatic cells of patients to treat disease. A defective gene is corrected by gene replacement, or the missing function is provided by the addition of a wild-type copy of the gene. For gene therapy to work, enough of the cells of the critically affected tissues or organs must be transformed with the functional copy of the gene to restore normal physiology.
Differentiate between genetic maps and physical maps
Genetic maps (also called linkage maps) provide a rough approximation of the locations of genes relative to the locations of other known genes, these maps are based on the genetic process of recombination Physical maps are based on the direct analysis of DNA, and they place genes in relation to distances measured in number of base pairs, kilo bases, or mega bases
How is a genome-wide association study carried out?
Genome-wide association studies survey numerous genetic polymorphisms scattered throughout the genome, particularly single-nucleotide polymorphisms (SNPs), for linkage to diseases or to other traits. Hundreds or thousands of individuals, some with the trait and some without, are each typed for hundreds of thousands of SNPs. SNPs that show strong statistical association to a particular trait indicate that a gene that contributes to the trait is located near these SNPs. Because SNPs across the entire genome are surveyed, genome-wide association studies can reveal multiple genes that contribute incrementally to complex, quantitative traits.
Explain how a gene library could be screened
Genomic and cDNA libraries can be screened with a probe to find the gene of interest -Take a membrane and gently tap to the colonies to take up a few cells from each colony, and treat the paper with something to lyse the cells to it, then use a probe. Comparison of the membrane with the master plate reveals which bacterial colonies have the DNA of interest
What are homologous sequences? What is the difference between orthologs and paralogs?
Homologous sequences are derived from a common ancestor. Orthologs are sequences in different species that are descended from a sequence in a common ancestral species. Paralogs are sequences in the same species that originated by duplication of an ancestral sequence and subsequently diverged. Paralogs may have diverged in function.
Describe several different methods for inferring the function of a gene by examining its DNA sequence.
Homology searches: For protein-coding genes, the DNA sequence provides information about the amino acid sequence of the protein it encodes. The amino acid sequence of the protein may yield clues to its function if it is similar to another protein of known function. For example, it is quite easy to recognize histones because their amino acid sequences are highly conserved among eukaryotes. Protein Domains: Even if the whole protein is not similar, it may have regions, or domains, consisting of a particular arrangement of amino acids with known functions or properties. Protein domains have been conserved over evolutionary time; there are a limited number of them, which have been mixed and matched through evolutionary time to produce the present-day protein diversity. Thus, an amino acid sequence from a newly sequenced genome can be scanned against a database (see Table 20.2 for examples) of known domains.
Describe how RNAi could be used for the purpose of reverse genetics
If we silence genes with RNAi, it temporarily turns off the gene and then we observe the phenotype
Briefly explain in situ hybridization
In situ hybridization, it involves hybridization of radiolabeled or fluorescently labeled DNA or RNA probes to DNA or RNA molecules that are still in the cell or part of intact chromosomes. This technique can be used to visualize the expression of specific mRNAs in different cells and tissues and the location of genes on metaphase or polytene chromosomes.
Briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence.
In the first step of PCR, the double-stranded template DNA is denatured by high temperature . In the second step, primers corresponding to the ends of the DNA sequence to be amplified are annealed to the single-stranded DNA template strands by lowering the temperature. In the third step, the temperature is raised to the optimal temperature for a thermostable DNA polymerase to extend from the primers so that the target DNA sequence is duplicated. These steps are repeated 20 times or more. Each cycle of denaturation, primer annealing, and extension results in doubling the number of copies of the target sequence between the primers
Define recombinant DNA technology
Is a set of molecular techniques for locating, isolating, altering, and studying DNA segments
Define comparative genomics
Is the field of genomics that studies similarities and differences in gene content, function, and organization among genomes of different organisms
Define structural genomics
Is the study of the organization and sequence of the genetic information contained within a genome, providing the basic DNA sequence information that is used in functional and evolutionary studies.
What are knockout mice, how are they produced, and for what are they used?
Knockout mice have a target gene disrupted or deleted ("knocked out"). First, the target gene is cloned. The middle portion of the gene is replaced with a selectable marker, typically the neo gene that confers resistance to the drug G418. This construct is then introduced back into mouse embryonic stem cells and cells with G418 resistance are selected. The surviving cells are screened for cells where the chromosomal copy of the target gene has been replaced with the neo-containing construct by homologous recombination of the flanking sequences. These embryonic stem cells are then injected into mouse blastocyst-stage embryos, and these chimeric embryos are transferred to the uterus of a pseudopregnant female mouse. The knockout cells will participate in the formation of many tissues in the mouse fetus, sometimes including germ-line cells. The chimeric offspring are mated to white mice (with the wild-type alleles) to determine whether the knockout gene is present in germline cells and transmitted to the next generation. The heterozygous offspring are interbred to produce offspring that are homozygous for the knockout allele. The phenotypes of the knockout mice provide information about the function of the gene.
Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used to determine which cells contain a particular plasmid.
Many plasmids designed as cloning vectors carry a gene for antibiotic resistance and the lacZ gene. The lacZ gene on the plasmid has been engineered to contain multiple unique restriction sites. Foreign DNAs are inserted into one of the unique restriction sites in the lacZ gene of plasmids, and the plasmids are transformed into E. coli cells lacking a functional lacZ gene. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid. The medium also contains an inducer for the lac operon, so the cells express the lacZ gene, and X-gal, a substrate for β-galactosidase that will turn blue when cleaved by β-galactosidase. The colonies that carry plasmid without foreign DNA inserts will have intact lacZ genes, make functional β-galactosidase, cleave X-gal, and turn blue. Colonies that carry plasmid with foreign DNA inserts will not make functional β-galactosidase because the lacZ gene is disrupted by the foreign DNA insert. These colonies will remain white. Thus, cells carrying plasmids with inserts will form white colonies.
Describe how next-generation sequencing works, specifically illuminated sequencing
Next generation sequencing - sequencing in parallel which means thousands/millions of DNA fragments simultaneously sequenced Illumina sequencing uses a special dNTP with a fluorescent tag so each base emits a unique fluorescent signal
Given the following measurements, which ones correlate to physical maps? Which to genetic maps? -cM - Physical distance - Recombination frequencies - More accurate - greater resolution - map units - lower resolution - base pairs - linkage analysis - less accurate
Physical Maps - base pairs - physical distance - greater resolution - more accurate Genetic maps - cM - recombination frequencies - map units - linkage analysis - lower resolution - less accurate
How is RNA interference used in the analysis of gene function?
RNA interference is a reverse-genetics approach to analyze gene function. A gene of interest is silenced by introducing siRNA complementary to the mRNA of that gene into a cell or organism. Recall that siRNAs are small RNA molecules that combine with proteins to form an RNA-induced silencing complex (RISC). The RISC pairs with complementary sequences on mRNA to either bring about degradation of the mRNA or inhibit its translation. Double-stranded RNA may be injected directly into a cell or organism, or the cell or organism may be genetically modified to express a double-stranded RNA molecule corresponding to the target gene.
How are RNA molecules sequenced?
RNA molecules are isolated from the cells of interest and then converted to cDNAs using reverse transcriptase. The cDNAs are prepared for sequencing by fragmenting them into smaller pieces, a few hundred nucleotides in length, then ligating adapters to the ends of the fragments. Primers anneal to the adapters for PCR amplification, followed by DNA sequencing.
Briefly describe the methods for inserting foreign DNA into plasmids that were described in this chapter.
Restriction enzyme cloning: - Vector and foreign DNA are cut with the same restriction enzyme and then ligated together with DNA ligase. This is the most straightforward, simplest method of cloning. The disadvantage is that matching restriction sites may not be available. Ligation may also produce undesirable products, such as the vector ligating to itself, without the foreign DNA insert. PCR fragment cloning: - DNA fragments generated by PCR may be ligated to plasmid vectors in either of two ways. One way is to synthesize PCR primers that have restriction sites at or near their 5′ ends. The resulting PCR fragments can be digested with the appropriate restriction enzymes to generate sticky ends for restriction cloning as described above. Another way is by addition of linkers: small synthetic DNA sequences containing restriction sites that are ligated to the blunt ends of DNA fragments. Restriction digestion then generates sticky ends that can be ligated as in restriction cloning. The advantage is that the method does not depend on the availability of suitable restriction sites; any restriction site can be added. The disadvantage is that this involves extra steps and is more difficult than simple restriction cloning.
What normal role do restriction enzymes play in bacteria? How do bacteria protect their own DNA from the action of restriction enzymes?
Restriction enzymes are used in bacterial cells as defense against viruses. These enzymes cut the viral DNA into fragments. Bacteria protect their own DNA by modifying bases, usually by methylation, at the recognition sites.
What is a single-nucleotide polymorphism (SNP)? How are SNPs used in genomic studies?
SNPs are single base-pair differences in the sequence of a particular region of DNA from one individual compared to another of the same species or population. SNPs are inherited as allelic variants and thus are useful as molecular markers for gene mapping and pedigree analysis. SNPs may themselves be associated with particular phenotypic differences, including genetic diseases. SNPs can also be used to study the function and evolution of genomes.
Define Single Nucleotide Polymorphisms and haplotype
Single Nucleotide Polymorphisms - single-base-pair differences in DNA sequences between individuals of a species - arise through mutation - most do not result in a different phenotype Haplotype: set of SNPs on a chromosome
Define - Southern Blot - Northern Blot - Western Blot
Southern Blot: transfer of DNA to membrane Northern Blot: transfer of RNA to membrane Western Blot: transfer of protein to membrane **These blots are then followed up with hybridization with a probe
Explain the steps of PCR - 3 steps
Step 1: A starting solution of DNA is heated to denature/break the hydrogen bonds between strands, producing single stranded templates Step 2: the DNA solution is cooled quickly, the DNA strands do not have a change to reanneal but the primers (which are short and single stranded) are able to attach to the template strands in their complementary sequences Solution 3: the solution is heated, DNA polymerase can synthesize new DNA strands and the entire cycle is repeated, each time the amount of target DNA doubles
Define recombinant DNA
Take DNA from 2 different sources and recombine the DNA (put DNA together from 2 different sources)
A molecular biologist wants to isolate a gene from a scorpion that encodes the deadly toxin found in its stinger, with the ultimate purpose of transferring this gene to bacteria and producing the toxin for use as a commercial pesticide. Isolating the gene requires a DNA library. Should the genetic engineer create a genomic library or a cDNA library? Explain your reasoning.
The genetic engineer should create a cDNA library from mRNA isolated from the scorpion venom gland. Bacteria cannot splice introns, and even if they did, the presence of introns may make the gene too large to insert the complete gene in a plasmid vector. If the engineer wants to express the toxin in bacteria, then the engineer needs a cDNA sequence that has been reverse-transcribed from mRNA, and therefore has no intron sequences. The venom gland must be the source of the mRNA for cDNA synthesis, so that the cDNA library will be enriched for toxin cDNAs. To have the mRNA and protein expressed, the cDNA should be cloned into an expression vector with sequences for transcription and translation in bacterial cells.
A geneticist uses a plasmid for cloning that has the lacZ gene and a gene that confers resistance to penicillin. The geneticist inserts a piece of foreign DNA into a restriction site that is located within the lacZ gene and uses the plasmid to transform bacteria. Explain how the geneticist can identify bacteria that contain a copy of a plasmid with the foreign DNA.
The geneticist should plate the bacteria on an agar medium containing penicillin to select for cells that have taken up the plasmid. The medium should also have X-gal and an inducer of the lac operon, such as IPTG or lactose. Cells that have taken up a plasmid without foreign DNA will have an intact lacZ gene, produce functional β-galactosidase, and cleave X-gal to make a blue dye. These colonies will turn blue. In contrast, cells that have taken up a plasmid containing foreign DNA inserted into the lacZ gene will be unable to make functional β-galactosidase. These colonies will be white and are the ones that the geneticist should choose from the plate.
What aspect of DNA structure causes DNA to migrate within an electrophoretic gel?
The negative charges on the phosphate groups of DNA.
What feature is commonly seen in the sequences recognized by restriction enzymes?
The recognition sequences are palindromic and are 4 to 8 base pairs long.
Describe the origin of restriction enzymes and how their names are derives
The restriction enzyme abbreviations come from the bacteria for example EcoRI - derives from E.coli - R strain - Enzyme #1
Will restriction sites for an enzyme that has 4 bp in its restriction site be closer together, farther apart, or similarly spaced, on average, compared with those of an enzyme that has 6 bp in its restriction site? Explain your reasoning.
The restriction sites for an enzyme with a 4-bp recognition sequence will be spaced closer together than the sites for an enzyme with a 6-bp recognition sequence. The 4-bp recognition sequence will occur with an average frequency of once every 4^4 = 256 bp, whereas the 6-bp recognition sequence will occur with an average frequency of once every 4^6 = 4096 bp.
Define epigenetics (Textbook)
The term epigenetics is based on the Greek root epi, meaning "over" or "above"; the term epigenetics has come to represent the inheritance of variation above and beyond differences in DNA sequence. Epigenetics usually refers to the phenotypes and processes that are transmitted to other cells and sometimes to future generations but that are not the result of differences in the DNA base sequence
You have discovered a gene in mice that is similar to a gene in yeast. How might you determine whether this gene is essential for development in mice?
The traditional method requires that the gene must first be cloned, possibly by using the yeast gene as a probe to screen a mouse genomic DNA library. The cloned gene is then used to create a knockout mouse strain. A neo gene is inserted into an exon of the cloned mouse gene. The cloning vector also includes the tk (thymidine kinase) gene, outside of the mouse gene region. This construct is then introduced into mouse embryonic stem cells. Selection of G418-resistant and ganciclovir-resistant cells will identify transformants where homologous recombination resulted in insertion of the knockout gene into the chromosome of the mouse embryonic stem cells. These knockout stem cells are injected into blastocyst stage embryos, which are then transferred to the uterus of a pseudopregnant mouse. Resulting chimeric progeny are tested for the presence of the knockout allele. Those that have the knockout allele in the germline are mated to wild-type mice to produce heterozygous progeny. Heterozygotes with the knockout allele are interbred. If the gene is essential for embryonic development, no homozygous knockout mice will be born. The arrested or spontaneously aborted fetuses can then be examined to determine how development has gone awry in fetuses that are homozygous for the knockout allele. Given that the mouse genome has been sequenced, one may now use the new CRISPR-Cas technology to edit the gene in the mouse genome. One would synthesize and micro-inject or transform sgRNA complementary to the target gene, along with Cas9 protein or a vector that expresses Cas9 protein, into a mouse egg or embryonic stem cell. The complex of sgRNA and Cas9 protein will cut the target DNA. Cells will repair the double-stranded DNA break via nonhomologous end joining, which typically introduces small insertions or deletions, causing frameshift mutations. PCR amplification of the target gene and DNA sequencing will verify which embryos or stem cell clones have frameshift mutations in the target gene. As for mice with a traditional knockout gene, heterozygous mouse lines are established and the heterozygotes are mated to obtain homozygotes for the frameshift mutation.
Much research has been devoted to developing methods for detecting, sequencing, and measuring levels of RNA (see Chapters 13 and 23) within the cell. All RNA is transcribed from DNA, so what information does RNA provide that is not found in DNA sequences? Give at least three examples.
There are two copies of each DNA sequence (for autosomes) in a cell, but the amount of RNA varies widely based on which genes are expressed and how much they are expressed. Thus, levels of RNA provide information on levels of gene expression. Samples of RNA from different developmental stages also may reveal that a gene is expressed only at particular times. Information on amounts and specific times of expression often provides insight into cell function. Also, RNA sequences can provide information on alternative splicing and other types of RNA modifications, which are not present in the DNA sequences.
Define heat map
To visualize the results, the scan of microarrays is often converted to a heat map. A heat map is a visual representation of a continuous variable, like the expression of a gene. The shade of the color indicates the value of the variable. EX. red might represent overexpression of the gene, green underexpression of the gene, and black average expression
Define DNA fingerprinting Know the type of sequence that is analyzed in DNA fingerprinting
Use DNA sequences for identification They look for micro satellites, which are tandem repeated sequences in noncoding regions. The number differs among individuals - aka: short tandem repeats (STR) - What we're doing when we do DNA fingerprinting is we're saying how many repeats does this person gave at this particular locus -Each STR located on different chromosome - Assort independently
Describe how restriction enzymes can be used to create recombinant DNA
We use restriction enzymes whenever we're making recombinant DNA - We cut DNA from the 2 different sources with the same restriction enzyme, then put them together in a tube, and the DNA pieces are going to come together
What is a microarray? How can it be used to obtain information about gene function?
a microarray consists of thousands of DNA fragments (probes) attached to a solid surface in an ordered grid (gene chips). The identity of the DNA probe at each location is known. Gene chips are typically used in hybridization experiments with labeled mRNAs or cDNAs to survey the levels of transcript for thousands of genes, or even whole genomes, at one time. The relative amounts of hybridization to each probe reveals sets of genes that are upregulated or down-regulated under defined environmental conditions, times of development or in disease states versus normal. Proteins or peptides may also be arrayed onto solid surfaces (protein chips), and can be used to identify protein-protein interactions or enzymatic activities or other properties of proteins
How are probes used to screen DNA libraries? Explain how a synthetic probe can be prepared when the protein product of a gene is known.
the DNA library must first be plated out, either as colonies for plasmid libraries or phage plaques on a bacterial lawn for phage libraries. The colonies or plaques are transferred to membrane filters, and their DNA is denatured and fixed to the filter. A labeled nucleic acid probe can be used to identify colonies or phage plaques that contain identical or similar DNA sequences. The probe hybridizes with DNA on the filter that has complementary sequences. Sometimes, cloned DNA from another organism can be used as a probe because successful hybridization does not require perfect complementarity. If no cloned DNA probe is available, but the amino acid sequence of the protein is known, then degenerate synthetic oligonucleotides can be synthesized that represent all possible coding sequences for a sequence of 7 to 10 amino acids. The mixture of synthetic oligonucleotides can be end-labeled and used as hybridization probes to screen a DNA library.
Define genomics
the branch of molecular biology concerned with the structure, function, evolution, and mapping of genomes.
Hybridization with a probe
• The method used to detect specific nucleotide sequence in an unknown sample by using a gene probe • Gene probe is a short segments of DNA of a known sequence • A probe carries a radioactive label Hybridization --> talking about is DNA and DNA/RNA coming together, complimentary base pairing. Polynucleotide strands hybridize together to make the double-helix
