Micro Exam 2

Steps of Gene Cloning

1. Isolation and fragmentation of source DNA (cut DNA with restriction enzyme) 2. Insertion of DNA fragment into cloning vector (add vector cut with same restriction enzyme) 3. Introduction of cloned DNA into host organism (add DNA ligase to form recombinant molecules)

What type of polymerase do we use for PCR?

A thermostable DNA polymerase is needed because of the high temperatures. -Taq or Pfu polymerase will be used

Why is a special polymerase, such as Taq polymerase, required for PCR? A. Taq polymerase can add DNA or RNA, allowing amplification of DNA or RNA. B. Taq polymerase can add complementary bases in an extremely accurate way, resulting in a very low error rate. C. Taq polymerase adds bases more rapidly than other polymerases, allowing very rapid amplification. D. Taq polymerase is produced by an extremophile prokaryote and is able to work at relatively high temperatures.

D

Pros and Cons of B. Subtilis as a host for cloning vector:

Pros: -Easily transformed -Nonpathogenic -Naturally secretes proteins -Endospore formation simplifies culture Cons: -genetically unstable -genetics less developed than e.coli

Pros and Cons of E. Coli as a host for cloning vector:

Pros: -well developed genetics -many strains available -most studied bacterium Cons: -potentially pathogenic -periplasm traps proteins

Pros and Cons of S. cerevisae as a host for cloning vector:

Pros: -well developed genetics -nonpathogenic -can process eukaryotic mRNAs -easy to grow Cons: -plasmids unstable -will not replicate most bacterial plasmids

Nucleic acid hybridization:

complementary base pairing of single strands of DNA/RNA from two different sources to give a hybrid double helix

Why is DNA polymerase from Thermus aquaticus ideal for PCR? A. It can synthesize DNA 5' to 3' and 3' to 5'. B. It does not require primers. C. It does not require energy to polymerize DNA. D. It can withstand the high temperatures associated with PCR.

D

Gel electrophoresis:

employs an agarose gel to separate nucleic acids by size and charge

Reporter gene:

encodes protein easy to detect and assay -may be used to report absence/presence of a genetic element or DNA in a vector -can be fused to other genes or other gene promoters to study gene expression

Heterologous expression:

expressing a gene in a different host

Protein fusions:

genes encoding two proteins are fused to share the same transcriptional and translational start and stop and yield one hybrid polypeptide

Southern blot:

hybridization procedure where DNA is the target and probe is RNA or DNA

Northern blot:

hybridization procedure where RNA is the target and probe is DNA or RNA

Gene disruption

inserts cassettes in middle of a gene, disrupting coding sequence -loss of function of gene in which cassette is inserted is a knockout mutation -gene knockouts determine whether a gene is essential

Molecular cloning:

movement of a gene from original source to small and manipulable genetic element (vector) -results in recombinant DNA -Gene can be manipulated -cloned DNA replicated

For gene cloning where can we get our DNA from?

polymerase chain reaction, DNA that's made by reverse transcriptase, or synthetic DNA made in vitro

Restriction endonucleases/enzymes:

recognize specific DNA sequences (restriction sites) and cut phosphodiester backbone, resulting in double stranded breaks -these recognition sequences are usually inverted repeats called palindromes -protect against viral DNA attack -cell protected from own restriction enzymes by chemical modification (usually methylation) -"sticky" short single stranded overhangs (EcoRI) or blunt ends (EcoRV)

A(n) ________ gene is a gene that encodes a protein that is easy to detect and assay. recorder translational encoder reporter

reporter

Nucleic acid probes:

segments of known single-stranded DNA used in hybridization

DNA cassettes

synthetic fragments that can mutate DNA via cassette mutagenesis

Viral pathogenesis

the study of the mechanism by which a virus causes disease

FISH (Fluorescent In Situ Hybridization)

uses fluorescent probes to target specific sequences in cells -identifies pathogens in clinical samples or bacteria in environmental samples

Genetic engineering:

using in vitro techniques to alter genes in the laboratory

Transformation/Oncogenesis:

virus induces ongoing cell division -may be productive or non-productive infection

Non-cytopathic infection

virus is produced without obvious cell damage -death and disease can still happen because immune system may kill cells or they may be inhibited

Steps of replication for viruses

1. Attachment (binding to the cell) 2. Penetration (entry into the cell) 3. Amplification (synthesis of viral proteins and genomes) 4. Assembly (of new virions, from individual proteins and genomes) 5. Release (of virions from the cell)

Gene fusions

- consist of segments from two different genes - promoters can be changed

Other adjustments for high-efficiency translation

-Codon usage: related to concentration of appropriate tRNA -Site directed mutagenesis can be used to change selected codons to match usage of host

Unenveloped viruses

-Don't have a lipid bilayer membrane -Capsid proteins bind to host cell receptors -Facilitate entry of the genome into the cell (capsid stays outside) -Major targets of the immune response

What does a capsid do?

-Package and stabilize the viral genome, which is very important for RNA -Protect viral genome in the environment

Steps of PCR:

1. Denature template DNA by heating and add two DNA oligonucleotide primers in excess (90-96 C) 2. DNA polymerase extends primers using template DNA 3. Heat to separate strands and cool (55-65 C) 4. Repeat 30 to 40 times to yield a million-billion fold increase

What are viruses?

-They are infectious agents -All have a genome surrounded by a protein coat -Obligate intracellular parasites -They assemble de novo (from nucleic acid and protein components) -They do not divide, they are not cells

Step 5: Release

-Upon cell death -Budding through plasma membrane (enveloped viruses) -Maturation: the virus particle may continue to mature after release from the cell

What do the host cells supply to viruses?

-amino acids -tRNA -Ribosomes -energy in the form of ATP -nucleotides and nucleosides

Hosts for cloning vectors

-are most useful if they are easy to grow and transform with engineered DNA -are genetically stable in culture -they must replicate the vector -helpful if background information and tools for manipulation exist -E. coli, B. subtilis, and S. cerevisae are usually used

T7 expression vectors

-cloned genes are placed under control of the T7 promoter -gene for T7 RNA polymerase present and under control of easily regulated system -BL21 E. Coli strains specially designed to work with pET T7 expression vectors and induction by IPTG

Disease

-direct damage to tissues or organs -damage caused by host immune responses (causes the most damage) -loss of critical cell functions -tumor development

Enveloped virus

-more fragile than non-enveloped viruses (they are damaged with alcohols and detergents) -facilitate entry of the genome into the cell (capsid stays outside) -targets of the immune response -easier to inactivate than non-enveloped viruses

Inapparent infection

-no disease and no clinical evidence of infection -a virus can still kill some hosts due to the host's genetics

Steps of RT-PCR:

1. Addition of primer and reverse transcriptase 2. Reverse transcription forms single-stranded cDNA 3. Addition of RNase 4. Addition of primer specific to 5' end plus Taq polymerase

What is the temperature used for the extension step? 72 °C 94 °C 60 °C

72

What is the sequence of the temperatures of a typical PCR reaction? 72 °C, 94 °C, 60 °C 60 °C, 72 °C, 94 °C 94 °C, 60 °C, 72 °C 72 °C, 60 °C, 94 °C 94 °C, 72 °C, 60 °C

94 °C, 60 °C, 72 °C

A polymerase chain reaction (PCR) copies an individual gene segment in vitro with a(n) ________ primer(s). A. pair of DNA B. individual RNA C. pair of RNA D. individual DNA

A

Step 4: Assembly

Assembly of viral proteins and nucleic acids into progeny particles

If an electrophoresis gel is run with RNA and then a DNA probe is used to identify the fragments of interest, what is the process called? A. Southern blotting B. Northern blotting C. Western blotting D. Eastern blotting

B

Restriction endonucleases are found in nature. They are extremely useful for genetic engineering. Why do organisms produce them? A. They are involved in DNA replication in prokaryotes. B. Because they cut only at specific sequences in DNA, they are useful in cutting harmful DNA (such as viral DNA) without harming the organism that produces them (which can protect those sequences in its own DNA). C. They are part of the viral life cycle and help in the assembly of new viruses. D. Organisms produce them as a way of allowing new genetic material to be inserted.

B

What is a thermocycler? A. The special DNA polymerase, used in a PCR reaction, that can tolerate the high temperatures B. The machine that controls the heat of the reaction, cycling between the different temperatures of the different steps during PCR C. The name for the DNA primers used in a PCR reaction D. The process of cycling through the different temperatures of a PCR reaction 30 times

B

What is the function of the primers in PCR? A. They are the monomer building blocks from which the DNA strand is synthesized. B. They provide a 3' end for the DNA polymerase. C. They polymerize free nucleotides to form the new DNA strands. D. They provide energy for the DNA polymerization reactions.

B

In which direction does DNA polymerase synthesize the new DNA strand? A. 3' to 5' B. Both 5' to 3' and 3' to 5' C. 5' to 3'

C

What's another word for protein coat?

Capsid

What does segmented mean?

Carries different pieces of nucleic acids (influenza is segmented and has 8 different nucleic acids)

What are the applications of PCR?

Cloning or sequencing, phylogenetic studies, amplifying very small DNA quantities, medical diagnostics, and forensic science

Electrophoresis can be used to separate molecules by size, shape, and charge. When DNA samples are run in an electrophoresis gel, the different bands produced generally represent fragments of different sizes. Why is the size of the fragment the most critical factor in determining how far it migrates on a gel when DNA fragments are compared? A. special type of gel is used for DNA electrophoresis to minimize the effects of charge. B. The charge on DNA is so small that it has a minimal effect on movement in the gel. C. The shape of the DNA fragments has an even greater effect on movement than size or charge, so charge is relatively unimportant. D. DNA moves toward a positive charge due to the negative charge on its phosphate groups. The charge is consistent because all DNA nucleotides have a single phosphate group rather than having more diverse patterns of charges. Because the charge is relatively consistent, size is the most important factor determining how far fragments move.

D

How do the strands separate during PCR? A. The primers separate the strands during the annealing step. B. The DNA polymerase breaks the hydrogen bonds between the two strands. C. The cycling of the temperatures breaks the hydrogen bonds between the two strands. D. The high heat of the denaturation step breaks the hydrogen bonds between the two strands.

D

Plasmids are commonly used as cloning vectors because __________. a. they are easily inserted into cells by transformation b. they can replicate independently of the chromosome c. they can contain genes for antibiotic resistance used for plasmid selection d. All of the listed responses are correct.

D

What provides the energy for DNA polymerization in a PCR reaction? A. Primers B. DNA polymerase C. Template DNA D. Deoxyribonucleoside triphosphates

D

What are the basic techniques of genetic engineering?

DNA amplification, electrophoresis, nucleic acid hybridization, molecular cloning, expressing foreign genes, and targeted mutagenesis

What does PCR require?

DNA polymerase and artificial oligonucleotide primers made of DNA

Polymerase Chain Reaction:

DNA replication in vitro, multiplying segments of target DNA up to a billionfold during amplification

Step 2: Penetration:

Entry of the genome into the cell and "liberation" of the viral nucleic acid. For enveloped viruses, penetration may occur via fusion at the cell membrane or after endocytosis

What are gels stained with so nucleic acids can fluoresce?

Ethidium bromide

T/F: Infection always equals disease

False

T/F: Primers are not homologous to gene of interest

False, primers are homologous to genes of interest

T/F: Cell death always causes disease in the infected host

False. Cell does not always cause disease in the infected host

T/F: Viral genomes are only made of RNA

False. The genomes can be of DNA or RNA

What was the earliest definition of viruses?

Filterable agents because they were smaller than bacteria, but this definition does not hold today. There are several DNA viruses who are as large as bacteria.

Baltimore classification

Groups viruses based on genome type and replication strategy Viruses with a DNA genome Viruses with an RNA genome Viruses that encode reverse transcriptase

Step 1: Attachment

Interaction of specific viral proteins with host-cell receptors. This is a lock and key type of mechanism.

Fusion proteins

Join target and carrier proteins to simplify purification -fuse two genes linked with a protease cleavage site into one coding sequence -transcription and translation yield a single protein -cleavage by protease releases target -carrier protein does not form inclusions

What does in vitro mean?

Life in a test tube

In DNA cloning, fragments of DNA need to be joined together (e.g., to add a fragment into a vector). What common enzyme is used for this process? helicases ligase DNA polymerase RNA polymerase

Ligase

Cloning the gene via mRNA or artificial synthesis

Must modify eukaryotic genes containing introns - typically modified via mRNA, which is easy to isolate because of polyA tails - convert into cDNA by RT-PCR lacking introns - bacterial promoters and RBS used for high-level expression -needs appropriate RBS and start codon

Are viruses alive?

No, a test tube of purified virus is metabolically inert. There is no ATP production, there is protein, DNA/RNA and some lipid. They also evolve

What is the main difference between DNA replication and PCR?

PCR uses DNA primers

What's a cloning vector?

Small, independently replicating genetic elements that can carry and replicate cloned DNA -designed to allow insertion of foreign DNA at a restriction site

Step 3: Amplification of viral proteins genomes (macromolecular synthesis):

Synthesis of the structural components (protein and nucleic acid) required to assemble new virions. -transcription of viral mRNA -Translation of viral proteins (protein synthesis) -genome replication

Reverse transcription PCR (RT-PCR)

These make DNA from an mRNA template, and they use reverse transcriptase to convert RNA into cDNA.

What are capsids?

They are assemblies of multiple copies of one of a few proteins. The protein subunits are capsomers. They are symmetrical and their two main shapes are icosahedral (spherical) and helical (rods)

How are viral genomes diverse?

They can be DNA or RNA, double or single stranded, circular or linear, segmented or unsegmented

How do nucleic acids migrate through the gel?

They migrate toward to the positive electrode because of their negatively charged phosphate groups -compared with a ladder (standard sample) -small molecules move faster than large molecules

How are viruses are grouped into Families?

This is based upon nucleic acid type / genome replication strategy, basic morphology, and presence/absence of an envelope

T/F: A virus can replicate in, and kill cells, but not cause disease

True

T/F: Conventional mutagens produce mutagens at random

True

T/F: During eclipse phase there is NO infectious virus

True

T/F: If source and vector have complementary sticky ends from being cut by same restriction enzyme, DNA ligase can anneal them

True

T/F: Only icosahedral viruses can be enveloped or non - enveloped

True

T/F: Recombinant proteins may cause problems due to degradation of proteases, toxicity, and insoluble inclusions

True

T/F: Expression vectors must ensure mRNA is efficiently translated

True -we need the appropriate ribosome-binding site (RBS) and start codon -bacterial RBSs must be engineered into vector for eukaryotic gene expression

Operon fusions:

coding sequence with its own translational start site and signals are fused to transcriptional signals of another gene

Site-directed mutagenesis

Uses synthetic DNA and cloning techniques to introduce mutations at precise sites -can change one or a few bases -can insert large DNA segments -requires oligonucleotides be available and chemically synthesized -allows any base pair to be changed -can test importance of amino acids

Quantitative PCR (qPCR):

Variation that quantifies initial amount of DNA

Cell death (cytopathic infection)

Virus blocks host cell transcription and translation Infected cell is killed by immune cells Cell undergoes apoptosis

What does unsegmented mean?

Virus carries only one piece of nucleic acid

Around what hour do the number of extracellular viruses particles appear?

after about 4.5 hours of infection

Expression vector:

allow experimenter to control the expression of cloned genes -this is important to regulate expression, especially at transcriptional level -require strong promoters

Thermocycler:

automated PCR machine

Which of the following sequences is a palindrome, characteristic of many recognition sequences for restriction endonucleases? a. GTAATG CATTAC b. GAATTC CTTAAG c. GGGGGGG CCCCCCCC d. TTGCCGA AACGGCT

b. GAATTC CTTAAG

Cloning vectors

can be of several types -use dependent on size of fragment to be cloned and host -plasmids are widely used

Reporter gene fusion:

coding sequence from reporter is fused with regulatory region from another source