Exam 5 Chapter 10

The classical approach to creating mutations is using chemical mutagens or radiation. However, new recombinant DNA technology can inactivate a known sequence using _________

RNAi (RNA interference)

Sequencing the human genome •A massive project •Sequencing methods in the late 1990s relied on the physical size of gels •Could only read a few hundred nucleotides at a time •Needed to sequence billions of nt pairs •Result: Does not represent the sequence of every individual's genome •Is the combined mosaic of a small number of anonymous donors, all of European origin •Other human genome sequencing has focused on different ethnic groups

Relied on the size of the sequence

A gene drive includes the editing machinery to affect the genome of

The first generation of offspring The second generation of offspring All generations of offspring Three of the above

In the horizontal gene transfer method of transformation, how is a plasmid introduced into a bacterial cell

Uptake from the environment surrounding the bacteria

In both Southern and Northern blotting techniques, what information can be obtained from the results? a. the size of the nucleic acid b. the abundance of the nucleic acid c. Both A and B d. the sequence of the nucleic acid

c

In creating a plasmid DNA vector, an antibiotic resistance gene is inserted to a. promote uptake of the plasmid by resistant bacteria b. allow bacterial division in the presence of antibiotic pressure c. select for bacteria that are transformed by a plasmid vector d. allow replication of the plasmid in both bacteria and target cells

c

Transgenic animals are produced by introducing altered gene material into embryonic stem cells. Mice carrying germ-line cells with the altered gene are mated with normal mice. The resulting offspring wil have a. the altered gene in germ line cells only. b. the altered gene in half of all cells. c. one copy of the altered gene in all cells. d. two copies of the altered gene in all cells.

c

What is the starting material for construction of a cDNA library? a. genomic DNA b. chromatin c. mRNA d. protein

c

Which library would you screen if the goal was to identify the coding sequence for a protein? a. genomic library b. RNA library c. cDNA library d. protein domain library

c

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c

Poverty can impact the genome by methylating over 1,500 genes affecting nearly _________ of human genes.

10%

From the normal to the amazing

1900 Normal Water closet (flushing toilet) Horse-drawn carriages Light bulb Amazing ØCars ØAirplanes ØAC 1950 Normal ØCars ØAirplanes ØAC Amazing ØCell phones ØComputers ØThe internet 2000 Normal ØCell phones ØComputers ØThe internet Amazing ØDesigner babies Ø? Ø?

When preparing a vector plasmid, what could you put in the multiple cloning site?

A target gene

When adding a gene of interest into an oocyte during genetic engineering, what is the preferred site of insertion?

A noncritical site

Which of the following represents "normal technology" in 1950?

Airplanes

As our species continues to advance in the technological capacity, the overriding concern for society will be the ________ of out progress

Ethics

What was the first requirement for developing the polymerase chain reaction (PCR)?

An isolated enzyme tolerant of high heat

After a genome has been sequenced it can be compared to similar sequences of other species using a program with the acronym

BLAST

The origin of replication in a DNA vector will allow replication of a plasmid in

Bacterial and target cells

Vitamin A deficiency can be responsible for causing

Blindness

When producing transgenic plants, bacteria may be used as a vector to carry a recombinant plasmid, what kind of cells will attract the vector so that the plasmid will be incorporated into a new plant shoot?

Callus cells

The value of using qPCR versus regular PCR is the ability to have

Can accurately measure the amount of mRNA

BACs—Bacterial artificial chromosomes •Definition: Engineered DNA molecules used to clone DNA sequences in bacterial cells Similar to plasmids except they can carry much larger pieces of DNA Vectors for Large-scale genome projects, Two high-capacity vectors for human genome project mapping mostly used yeast artificial chromosome (YAC), accepts million base pairs Sequencing used bacterial artificial chromosomes (BAC), accepts about 300,000 bp BACs are more stable, easier to work with than YACs

Can carry more YACs use yeast chromosomes

Which of the following would have been considered amazing in 1900?

Cars

CRISPR-Cas can be used to ___________ allow expression of the FMR1 gene that is silenced in Fragile X syndrome.

Change the FMR1 Promoter to a euchromatin state

Restriction enzymes from bacteria that cut DNA sequences at specific cleavage sites are very useful for

Cloning Making a DNA library Recombinant DNA for Transformation All of the above

To detect a viral genome in an individual, the first step is

Collecting a blood sample Collecting a fluid sample Two of the above

Mapping the genome to a physical genome map •A sequence-tagged site (or STS) is a short (200 to 500 base pair) DNA sequence that has a single occurrence in the genome and whose location and base sequence are known. Restriction map of one segment of human genome, restriction pattern for individual BAC clones, cleavage sites for restriction nucleases A, B, C, D, and E

Could not prove to be exact

As our species continues to produce enormous amounts of data, one of the most compact storage devices will be

DNA

Northern blotting •Method using the same techniques as Southern blotting but detecting RNA molecules instead of DNA •What could be used as a probe for RNA? (RNA nucleotides) If you have the RNA you will see a band Vero, H1299, rRNA, ATF4,

Detects RNA instead of DNA

CRISPR-Cas in bacteria •Employs two non-coding RNAs •trRNAs (tracer RNAs) •crRNAS (complementary sequences to trRNAs) •Spacer regions (color-coded) that will base pair with target sequences (each different based on viral seq to eliminate) •The crRNA-trRNA complexes with the Cas protein, an endonuclease trRNAs, crRNAs, The Cas9 gene is also expressed. Each tracrRNA c-RNA complex binds to a Cas9 protein (only one is shown), Cas9

EXPLANATION Non-Coding = Do not code for proteins

What class of protein is the Cas enzyme?

Endonuclease

To detect aneuploidy during a prenatal test, the preferred method is

FISH

CRISPR-Cas technology will be useful for the following

Fighting cancer Creation of BIofuel Eliminating genetic disease All of the above

Where is the PAM (Protospacer Adjacent Motif) located in the CRISPR-Cas Sequence?

Following the viral insert sequence

CRISPR is made of short palindromic repeats. What are the non-repetitive spacers between them?

Fragments of bacteriophage, prophages, and plasmids

In our microarray example, cells were tested under two conditions: Untreated or treated with a hormone. The results indicated the _________ of the cells

Gene expression

In order to produce exponential quantities of a target sequence via PCR, automatic pairing of nucleotides must occur due to

Hybridization

A cDNA (copy DNA) library is different from a whole genome library in that it does not contain

Introns

Oilseed crops that are developed to produce _______ oils help prevent overharvesting of salmon.

Omega 3

To determine organisms that may be associated with an unknown pathogen in the search for determining its identity, which of the following types of technology would you first use with a blood or soil sample?

PCR

The following technology is useful in detecting an RNA based viral genome

Polymerase chain reaction Gel electrophoresis Two of the above

Trade-offs Genetically modified crops and foods Projected Advantages May need less fertilizer, pesticides, and water Can be resistant to insects, disease, and drought Can grow faster May tolerate higher levels of herbicides Projected Disadvantages Have unpredictable genetic and ecological effects May put toxins in food Can promote pesticide-resistant insects, herbicide-resistant weeds, and plant diseases COuld disrupt seed market and reduce biodiversity

Round-Off Glycosate causes non-hodgkins lymphoma

Living robots have been engineered from stem cells. If they become damaged, they may _______ _____

Self repair

Caveats Pfizer vaccine requires two doses, 21 days apart Must be stored at -70 degrees C (-94 degrees F) Vaccines must treat 7.8 billion people Distribution Distribution to the rural areas? People are not near hospitals Native American population

Storing protein coding Eliminating PCR Gel electrophores Blood Fluid sample To the 3' hydro In a 5'-3' Only one at a time``

A benefit of using mRNA instead of whole genome DNA for genome libraries is

Storing protein-coding regions only Eliminating introns Two of the above

To edit a gene with a mutation, what kind of sequence can be inserted using CRISPR-Cas?

Synthetically produced Wild type Two of the above

Why must a DNA sample be cooled to 60 degrees C during PCR?

To allow nucleotides to anneal to the target sequence

Sanger sequencing is a method that takes advantage of the limitation of DNA construction that requires adding nucleotides

To the 3' Hydroxyl In a 5' to 3' direction Only at one time Three of the above

Reproducing the clone •During transformation, not all bacteria will take up the plasmid. •The researcher only wants to grow bacteria with the plasmid. •Solution? Plasmid replication inside bacteria Bacterial cell, double-stranded recombinant plasmid DNA introduced into bacterial cell, transformation, cell culture produces hundreds of millions of new bacteria, many copies of purified isolated from lysed bacteria

Transformation outside in liquid environment Cloning is a massive for isolating apiculus sequence of DNA from a complex mixture of different DNA sequences in order to clone the fragment of DNA we need to insert it into a vector usually a highly modified page or cloud I can replicate the host soon generally a plasmid vector contains 3 elements cloning site where the foreign DNA fragmente can be inserted drug resistance gene which destroys antibiotics in this case ampicillin to allow select growth of the host cell and a replication origin to allow the plasmid to replicate in the wholesale to insert foreign DNA we must first use a restriction enzyme to cleave the vector at the cloning site here equal or one please the palindromic sequence GAA TTC to produce single stranded ends all sticky ends these can hybridize with any piece of DNA that has also been tagged with eagle or one orange DNA containing the sequence we wish to clone digested with cheap or one and then mixed with the gleevec picky answer the form and plasma DNA molecules hybridize and then our steel into possible test or languages by the enzyme DNA ligase creating are common plastic each of these recombinant plasmids contains the inserted DNA fragmente and ampicillin resistance gene and a replication origin school or library of circular recombinant plasmids is thus created each plasmid carries a unique fragment of foreign DNA as indicated by colored arrows at the host cells coli bacteria are added to the recombinant plasmids tell have been treated with calcium chloride but make them permeable to DNA molecules through process called transformation few cells take up a recombinant plasmid most other cells do not bacterial cells are poured onto a plate of nutrient auger containing the antibiotic ampicillin with ampicillin in the auger only cells resistant to the drug and grow 37 degrees the cells will grow and multiply 'cause they cannot move on the auger each will produce a separate colony of cells last transform cells lack the plasmid with the ampicillin resistance gene and died cells that have a plasmid are resistant to ampicillin and survive the replication origin allows the plasma to replicate by using the host cells and times lastman replication is independent of host cell division the plasmids are distributed to each daughter cell when the host cell divides of the plasmids replicate the host cells multiply the number of copies of the recombinant plasmid is greatly amplified multiple daughter cells form a colony or clone because all the hotels in a colony are derived from a single still they all contain copies of the same recombinant plasmids with this fragment of born DNA variety of assay methods can now be used on the bacterial colonies to determine which contain particular DNA sequence we wish to isolate

Crossbreeding plants has been practiced for centuries. Selecting plants with the most favored traits for crossing is using a form of

Two of the above Artificial selection Hybridization

Comparative genome analyses So we have a sequence... What does it mean? What do we do next? ...we make comparisons...

`

Gene drive effect ...changes a heterozygote ...into a homozygote expressing a dominant trait

`

Quantitative Real-time PCR (qPCR) •Definition: Technique of collecting data throughout the polymerase chain reaction as it occurs. •Very recent: Became available in the 1990s Revolutionary: Accurate quantification of mRNA versus approximate measurements resulting from standard PCR and electrophoresis gels Top, direction of migration, bottom

``

In creating a genomic DNA library, what is the FIRST step? a. Fragmenting the DNA into manageable pieces b. Screening clones c. Transforming recombinant DNA into bacteria d. Ligating DNA into plasmids

a

What distinguishes qPCR from regular PCR? a. data is collected as the sample is processed b. reverse transcriptase enzymes do not need to be heat-stable c. use of both RNA and DNA as templates d. quantification provides approximate measurements

a

What enzyme is first required to amplify RNA via PCR? a. Reverse transcriptase b. Taq polymerase c. Ligase d. Poly A polymerase

a

What enzyme must be used first to create a cDNA library? a. Reverse transcriptase b. DNA ligase c. DNA polymerase d. RNA polymerase

a

Where are DNA libraries stored? a. in bacteria b. on shelves in large buildings c. in millions of tubes d. in computers

a.

Reporter genes are constructed in order to determine a. cell types that express a gene b. transcription factors that activate a gene c. regulatory sequences that affect gene activation d. the coding sequence of a gene similar to the reporter gene

a`

Cancer cell genome sequencing is a valuable tool in order to identify a. the passenger mutations that contribute to the cancer phenotype b. cancer cells that carry driver mutations c. early clonal expansion that precedes benign tumor growth d. chemotherapy resistant genes

b

In a typical PCR cycle, what is the first step? a. primer annealing b. denaturation/melting c. extension/polymerization d. ligation

b

Sometimes artificial chromosomes (AC) constructed from bacterial (BACs) or yeast (YACs) DNA are used to harbor genomic DNA for libraries (e.g., the human genome project). Why are BACs/YACs often used instead of traditional plasmid DNA for the construction of genomic libraries? a. genomic plasmid DNA cannot be efficiently ligated b. Typical plasmid DNA vectors can only harbor DNA up to a certain size (~3,000 bp) c. BACs/YACs are easier to store in DNA libraries d. Larger genomic DNA fragments in BACS/YACS have the introns removed

b

Southern blotting is a hybridization-based technique to measure a. RNA b. DNA c. Protein d. DNA-binding proteins

b

The specific site in a cell where a particular RNA is made can be detected by a. microarray. b. in situ hybridization c. RNA-seq d. RNAi

b

How does one find a book (gene/clone) of interest in a DNA library? a. by doing PCR on the whole pool of DNA b. by sequencing each clone in the library c. by next-generation sequencing d. by hybridizing a radiolabeled probe of the sequence to bacterial colonies

d

In most nucleic acid hybridization assays, the first step must be denaturation of double-stranded DNA. How can this be achieved? a. By heat b. by alkaline lysis c. by treatment with DNase 1 d. Both a and b

d

Second-generation DNA sequencing differs from early gel-processed sequencing due to the following: a. It does not require fluorescent-based dideoxynucleotides b. single strands of DNA are processed at high speeds c. it can process large genomes with shot-gun sequencing d. a chemical blocker is removed prior to ongoing strand synthesis

d

The CRISPR-cas gene editing technology is capable of editing a gene by removing or inserting a sequence. To rejoin strands after processing, CRISPR uses a. non-homologous end joining b. a spliceosome with ligase c. homologous recombination d. A and C

d

Which of the following is NOT a molecular biology technique in which PCR plays a role? a. DNA library amplification b. Clinical pathogen diagnostics c. STR analysis d. All of the above involve PCR

d

Which of the following is a technique to fragment DNA? a. Add a restriction enzyme to the purified DNA b. Passing the DNA repeatedly through a hypodermic needle to shear it c. Sonicate the DNA using high frequency vibration waves to break the phosophodiester bonds d. All listed methods can fragment DNA

d

Which of the following is true about DNA/RNA gel electrophoresis? a. nucleic acids migrate from the negative to the positive electrode b. larger nucleic acids remain near the top portion of the gel c. smaller nucleic acids remain near the top of the gel d. A and B

d

To explore the function of a gene in an organism that already has a sequenced genome, the following technology can be used a. RNAi b. microarray c. BLAST program on the NCBI website d. both A and C

d.

In order to study the function of a normal gene, researchers can use CRISPR-Cas for

gene knock out gene silencing Two of the above

Medical applications of cloned plasmids •Treatment for: •Diabetes •Cystic fibrosis •Severe combined immunodeficiency disease (SCID) •Other gene therapy How can the gene insert be retrieved? Top, direction of migration, bottom,

insulin made in bacteria

The template for making cDNA is

mRNA

The Southern blot (Gel-transfer hybridization) DNA Probe: short, single-stranded sequence complementary to the sequence of interest Ex. ACGCTAATGTAG The probe will be labeled (radioactive label common) Unlabeled DNA cut with.a restriction nuclease, electophoresis, labeled DNA of known sizes as size markers, agarose gel, stack of paper towels, nitrocellulose paper, buffer, drawn toward paper towels, carries alkali-denatured DNA fragments from the gel to the nitrocellulose, A. Double-stranded DNA fragments separated by agarose gel electrophoresis, Nitrocellulose paper carefully removed, Labeled DNA probe hybridized to the nitrocellulose-bound DNA, labeled DNA probe in buffer, sealed plastic bag, positions of pre-labeled markers, band labeled by probe, Labeled DNA probe hybridized to complementary DNA bands visualized by Autoradiography

Single-stranded DNA fragments blotted onto paper, using an alkali solution, less H+, separate DNA fragments, carries denatured protein up ijto the paper, the nitrocellulose paper is removed

Hybridization for DNA cloning •DNA cloning: Production of many identical copies of a DNA sequence 1.Classical approach to making recombinant DNA molecules → Enzyme required: DNA ligase Research value: any two pieces of DNA can be joined together in a test tube Vector: the recombinant DNA Circular double-stranded plasmid DNA (cloning vector), cleavage with restriction nuclease, DNA fragment to be cloned, covalent linkage by DNA ligase, recombinant DNA

Used in research DNA ligase required to ligase back together A plasmid is a cloning vector Any DNA can be added

Why must a DNA sample be heated to 90 degrees C before a target sequence can be copied via PCR?

To break hydrogen bonds

An alkali solution can denature DNA because it affects the

hydrogen bonds

Making cDNA 1.Extract mRNA from a cell 2.Use reverse transcriptase to transcribe DNA from mRNA 3.Transcribe a complementary strand of DNA using DNA polymerase cDNA from mRNA DNA in nucleus mRNAs in cytoplasm Cells in culture, Lyse cells and purify mRNA, mRNA, mRNA, oligo(dT) primer,1. incubate with reverse transcriptase to synthesize cDNA strand, mRNA, cDNA, 2. When cDNA strand is completed, hydrolyze RNA strand, cDNA, 3. Incubate with DNA polymerase to synthesize second DNA strand, S1 nuclease cuts loop, Double-stranded DNA, Double-stranded cDNA ready for insertion into appropriate cloning vector

mRNA makes cDNA Thymine added to make cDNA

Cloning a protein-coding gene •Many genes are too large to clone easily •Bacteria and yeast cannot remove introns from mammalian RNA •Solution: use cDNA instead The Factor VIII gene functions in blood clotting. Mutations are responsible for the most prevalent form of hemophilia. Human-beta-globin gene, human factor VIII, introns, exons, 200,000 nucleotide pairs, 2000 nucleotide pairs

26 exons, bacterias cannot remove introns, use cDNA

Preparation of genomic library Gene A Gene B, exon, intron, Gene B, nontranscribed DNA, chromosomal DNA, Restriction nuclease digestion to produce DNA fragments, DNA fragments, DNA cloning, Genomic DNA library, Preparation of cDNA library, Gene A, Gene B, transcription, RNA transcripts, RNA splicing, mRNAs, Treatment with reverse transcriptase and DNA polymerase to produce cDNA copies of mRNAs --> CDNA fragments, DNA cloning ---cDNA library

A Messenger RNA molecule is the three prime palie a tail so using an oligo DD primer and reverse transcriptase complementary strand of DNA for C DNA is synthesized forming an M RNA DNA hybrid The M RNA string is degraded with alkali and the free three prime end of the DNA strand forms a hairpin loop prime synthesis for second DNA strand using DNA polymerase 3 at one nuclease leaves the hairpin loop leaving a double strand in CD and a copy of the original M RNA

Probing a cDNA library Autoradiography: A technique using X-ray film to visualize molecules or fragments of molecules that have been radioactively labeled. Disc of absorbent paper, petri dish with colonies of bacteria containing recombinant plasmids, peel paper from dish to produce replica of colonies, DNA bound to paper, lyse bacteria and denature DNA with alkali, Radioactively labeled DNA probe,Incubate with probe and wash, colonies containing plasmid of interest, expose paper to photographic film, film, position of desired colonies detected by autoradiography

DNA polymerase 3, For a particular gene Denature DNA with alkali

Finding DNA sequence of interest 1.DNA denaturation: Separation of the two strands, usually with heat (~ 90 degrees C; nearly at boiling pt.) a)Hydrogen bonds release b)Covalent bonds do not 2.Hybridization, AKA DNA renaturation: Natural re-forming of the double helix. The process is driven by reformation of H-bonds. DNA double helices, heat, denaturation to single strands (hydrogen bonds between nucleotide pairs broken, slowly cool ---> Renaturation restores DNA double helices (nucleotide pairs re-formed)

Heat denatures DNA double helixes, breaks hydrogen bonds , covalent bonds do not break due to heat Hybridization, bringing together base pairs through hydrogen bonds, In the lab, bonding of one DNA sequence to a complementary DNA

Electrophoresis results •Two restriction enzymes to cut the same DNA genome •Use of more than one restriction enzyme allows user to determine where fragments are located •Limitation: will not reveal a sequence of interest •Next step: using a DNA probe DNA size markers, Double-stranded DNA, Cut with HindIII, Cut with EcoRI, Load DNA onto Gel and apply voltage, negative electrode, nucleotide pairs (*1000), 23, 9, 6.5, 4.3, 2.3, 2, positive electrode (A) +, Slab of agarose gel, Top ---> Direction of migration --> bottom

Restriction enzymes cut DNA genome DNA has negative charge, will run to the positive electrode