Docsity
Docsity
Prepare for your exams
Prepare for your exams

Study with the several resources on Docsity

Earn points to download
Earn points to download

Earn points by helping other students or get them with a premium plan

Guidelines and tips
Guidelines and tips
Sell on Docsity
Sell on Docsity
Docsity AI
Docsity AI
Log inSign up

Prepare for your exams

Study with the several resources on Docsity

Find documents

Prepare for your exams with the study notes shared by other students like you on Docsity

Search for your university

Find the specific documents for your university's exams

Docsity AINEW

Summarize your documents, ask them questions, convert them into quizzes and concept maps

Explore questions

Clear up your doubts by reading the answers to questions asked by your fellow students

Earn points to download

Earn points by helping other students or get them with a premium plan

Share documents
download point icon20 Points

For each uploaded document

Answer questions
download point icon5 Points

For each given answer (max 1 per day)

All the ways to get free points
Get points immediately

Choose a premium plan with all the points you need

Study Opportunities

Choose your next study program

Get in touch with the best universities in the world. Search through thousands of universities and official partners

Community

Ask the community

Ask the community for help and clear up your study doubts

Free resources

Our save-the-student-ebooks!

Download our free guides on studying techniques, anxiety management strategies, and thesis advice from Docsity tutors

Genetic Engineering Techniques and Cloning Vectors, Cheat Sheet of Microbiology

Busan National University of Education – BusanMicrobiology

Various aspects of genetic engineering and cloning techniques, including the use of different types of vectors, restriction enzymes, and molecular cloning procedures. It discusses the characteristics of an ideal cloning host, the importance of expression vectors for expressing eukaryotic genes in prokaryotes, the creation of dna libraries, the use of artificial chromosome vectors like bacs and yacs, the benefits of using t7 promoters and polylinkers in vectors, the process of site-directed mutagenesis, and the applications of gene fusion techniques like operon and protein fusions. A comprehensive overview of the fundamental concepts and techniques in the field of genetic engineering, making it a valuable resource for students and researchers studying molecular biology, biotechnology, and related disciplines.

Typology: Cheat Sheet

2019/2020

Uploaded on 06/13/2024

1-c5u
1-c5u 🇰🇷

1 / 11

Toggle sidebar

This page cannot be seen from the preview

Don't miss anything!

bg1
MULTIPLE CHOICE. Choose the one alternative that best completes the statement or
answers the question.
1) If a foreign gene is cloned into a host, it is important that the cloned host
itself
1) _______
A) repress the genetic expression being studied.
B) produce the protein in larger amounts than the vector.
C) not produce the protein being studied.
D) produce signal proteins to tag the host protein.
2) Detecting a specific protein with an antibody is considered a(n)
________ method.
2) _______
A) duplication B) isolation
C) selection D) screening
3) A vector that can be used to clone a desired gene that contains
regulatory sequences so the expression of the gene can be manipulated
is called a(n) ________ vector.
3) _______
A) stability B) endogenous
C) expression D) regulatory
4) Expression vectors are designed to ensure that ________ can be
efficiently ________.
4) _______
A) mRNA / translated B) DNA / transcribed
C) DNA / translated D) mRNA / transcribed
5) A(n) ________ gene is a gene that encodes a protein that is easy to detect
and assay.
5) _______
A) reporter B) encoder
C) translational D) recorder
6) One of the more formidable obstacles to mammalian gene cloning is the
presence of
6) _______
A) repressors. B) integrators.
C) introns. D) exons.
7) Which is the organism of choice for most molecular cloning? 7) _______
A) Bacillus subtilis B) Vibrio natriegens
C) Saccharomyces cerevisiae D) Escherichia coli
8) What type of vector can replicate and be maintained stably in two (or
more) unrelated host organisms?
8) _______
A) integrating B) shuttle
C) expression D) virus
9) The genes encoding luciferase, green fluorescent protein (GFP), and
β-galactosidase are typically used in cloning as
9) _______
A) reporter genes. B) transcription regulators.
C) global control genes. D) promoter sequences.
10) mRNA transcripts are converted into cDNA by 10) ______
A) DNA polymerase. B) RNA polymerase.
pf3
pf4
pf5
pf8
pf9
pfa

Partial preview of the text

Download Genetic Engineering Techniques and Cloning Vectors and more Cheat Sheet Microbiology in PDF only on Docsity!

MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question.

  1. If a foreign gene is cloned into a host, it is important that the cloned host itself

1) _______

A) repress the genetic expression being studied. B) produce the protein in larger amounts than the vector. C) not produce the protein being studied. D) produce signal proteins to tag the host protein.

  1. Detecting a specific protein with an antibody is considered a(n) ________ method.

2) _______

A) duplication B) isolation C) selection D) screening

  1. A vector that can be used to clone a desired gene that contains regulatory sequences so the expression of the gene can be manipulated is called a(n) ________ vector.

3) _______

A) stability B) endogenous C) expression D) regulatory

  1. Expression vectors are designed to ensure that ________ can be efficiently ________.

4) _______

A) mRNA / translated B) DNA / transcribed C) DNA / translated D) mRNA / transcribed

  1. A(n) ________ gene is a gene that encodes a protein that is easy to detect and assay.

5) _______

A) reporter B) encoder C) translational D) recorder

  1. One of the more formidable obstacles to mammalian gene cloning is the presence of

6) _______

A) repressors. B) integrators. C) introns. D) exons.

  1. Which is the organism of choice for most molecular cloning? 7) _______ A) Bacillus subtilis B) Vibrio natriegens C) Saccharomyces cerevisiae D) Escherichia coli

  2. What type of vector can replicate and be maintained stably in two (or more) unrelated host organisms?

8) _______

A) integrating B) shuttle C) expression D) virus

  1. The genes encoding luciferase, green fluorescent protein (GFP), and β-galactosidase are typically used in cloning as

9) _______

A) reporter genes. B) transcription regulators. C) global control genes. D) promoter sequences.

  1. mRNA transcripts are converted into cDNA by 10) ______ A) DNA polymerase. B) RNA polymerase.

C) reverse transcriptase. D) a restriction endonuclease.

  1. By definition, genetic engineering techniques are performed 11) ______ A) in situ. B) in toto. C) in vitro. D) in vivo.

  2. The class of restriction endonucleases that cleave the DNA within their recognition sequences is known as

12) ______

A) type III. B) type II. C) type I. D) All restriction endonucleases cut within their recognition sequence.

  1. Which of the following sequences is a palindrome? 13) ______ A) GGGGGGG CCCCCCCC

B) GAATTC

CTTAAG

C) TTGCCGA

AACGGCT

D) GTAATG

CATTAC

  1. Nucleic acid hybridization is widely used for ________ segments of DNA.

14) ______

A) characterizing B) identifying C) detecting D) all of the above.

  1. Molecular cloning includes 15) ______ A) obtaining and purifying a copy of the gene of interest. B) placing the gene of interest into a convenient vector. C) locating the gene of interest. D) all of the above.

  2. M13 is an ssDNA filamentous phage used for 16) ______ A) assembling vectors. B) hybridization. C) site-directed mutagenesis. D) creating gene fusions.

  3. To make an adequate library for sequencing prokaryotic genes, a vector should hold inserts in the range of

17) ______

A) 2 - 10 kbp. B) 10 - 50 kbp. C) 50 - 100 kbp. D) 0.5-l kbp.

  1. Bacterial artificial chromosomes (BACs) can be constructed using the 18) ______ A) M13 phage. B) R plasmid. C) M plasmid. D) plasmid.

  2. Which statement is TRUE? 19) ______ A) YACs and BACs undergo recombination and rearrangement at about the same rate. B) BACs are more likely than YACs to undergo recombination and rearrangement. C) YACs are more likely than BACs to undergo recombination and rearrangement. D) It is impossible to state with any certainty whether YACs or BACs are more likely to undergo recombination and rearrangement, because environmental factors play a major role in the probability

C) Northern blot D) Western blot

  1. Which of the following terms is used to describe a synthetic DNA fragment?

29) ______

A) DNA cassette B) DNA probe C) DNA hybrid D) operon

  1. Which construct would be MOST useful in studying translational control?

30) ______

A) operon fusion B) protein fusion C) gene fusion D) all of the above

TRUE/FALSE. Write 'T' if the statement is true and 'F' if the statement is false.

  1. Genetic engineering includes techniques that result in genetically modified organisms.

31) ______

  1. Often organisms used as hosts for genetic engineering must have specific genotypes to be effective.

32) ______

  1. Although a gene library may contain tens of thousands of clones, finding the clone of interest is the biggest challenge.

33) ______

  1. Strong promoters used for genetic manipulation are usually regulated by specific molecules.

34) ______

  1. It is usually desirable to design a vector that transcribes cloned genes at high levels at all times.

35) ______

  1. Although various codons often code for the same amino acid, it is important to choose the codon preferred by the expression host itself.

36) ______

  1. Viral vectors can be used for molecular cloning, but plasmid vectors cannot.

37) ______

  1. One problem with both BACs and YACs is that genetic regions of these chromosomes cannot be subcloned.

38) ______

  1. In principle, a type II restriction endonuclease with an 8-nucleotide recognition sequence should cut 1 in every 48 nucleotide positions.

39) ______

  1. One basic technique of genetic engineering includes the ability to cut the DNA of interest into random fragments.

40) ______

  1. Modification enzymes typically methylate specific bases within the recognition sequence to prevent digestion of the nucleotide sequence by restriction endonucleases.

41) ______

  1. Banding patterns generated from restriction analyses of either whole chromosomes or specific genes from a series of organisms can yield an indication of their genetic relationships.

42) ______

  1. Cloning vectors are small, independently replicating genetic elements use d to

replicate cloned genes.

43) ___

___

  1. One important advantage of eukaryotic cells as hosts for cloning vectors is that they already possess the complex RNA and posttranslational processing systems required for the production of eukaryotic proteins.

44) ______

  1. Site-directed mutagenesis is an in vitro method that removes mutations at specific locations in a sequence.

45) ______

SHORT ANSWER. Write the word or phrase that best completes each statement or answers the question.

  1. Artificial chromosomes are vectors capable of carrying ________. 46) _____________

  2. A(n) ________ uses a DNA or RNA probe to detect the presence of a particular DNA sequence. A(n) ________ uses a DNA or RNA probe that is used to detect the presence of a particular RNA sequence.

47) _____________

  1. Codon bias offers clues about reading frame functionality because most organisms show preferences among ________.

48) _____________

  1. The host for a BAC is typically a mutant strain of Escherichia coli that is missing ________ and is defective in ________.

49) _____________

  1. After DNA has been electrophoresed through a(n) ________, a stain such as ethidium bromide is used to visualize different DNA sequence ________.

50) _____________

  1. The process by which a fragment of DNA is placed into a vector where it can be replicated is called ________.

51) _____________

  1. A major disadvantage of using Bacillus subtilis as a cloning host is that over several generations of subculturing, ________.

52) _____________

  1. Two problems of using Escherichia coli as a cloning host are ________ and ________.

53) _____________

  1. One limitation to designing an antibody for detecting a particular protein is that the antibody must be ________ for the protein of interest.

54) _____________

  1. If the gene in question is not expressed and no assay or antibody is available to detect the gene product, the most common procedure used for identification is a(n) ________ probe.

55) _____________

  1. The ________ is a naturally occurring plasmid within yeast cells, and development of cloning yeast vectors is often based on this particular plasmid.

56) _____________

  1. Discuss why codon usage must be considered when cloning a gene into another organism.

  2. When trying to express a mammalian gene in a bacterium, expression vectors are often used. Using your knowledge of prokaryotic and eukaryotic genetics, explain why expression vectors are so important.

  3. Explain how a DNA library is created.

  4. Explain how foreign DNA of greater than 300 kbp can be inserted and stably maintained in BAC vectors.

  5. Explain what is necessary for a YAC to function as a normal eukaryotic chromosome.

  6. Describe a method developed by molecular biologists to easily observe the success of a genetic engineering procedure involving the ligating of a gene of interest into a plasmid.

  7. Explain how restriction sites, which are not random, can be a part of an approach to random cloning.

  8. Explain the function and usefulness of a polylinker within a vector.

  9. Describe the three main steps to clone a gene into an organism.

  10. Explain why DNA fragments migrate toward the positive electrode and why some fragments migrate more rapidly than others during gel electrophoresis.

  11. When is it appropriate to use an artificial chromosome vector? Describe a specific example.

  12. Explain how the process of cloning differs when a vector with sticky ends is used and when a vector with blunt ends is used.

  13. Describe the usefulness of blue-white screening, also called α-complementation, in cloning vectors such as pUC19. Include in your answer the terms polylinker, DNA ligase, lacZ gene, insertional inactivation, Xgal, and β-galactosidase.

  14. Explain the process of site-directed mutagenesis, and discuss some applications of this technique.

  15. Compare and contrast operon and protein fusions.

1) C

2) D

3) C

4) D

5) A

6) C

7) D

8) B

9) A

10) C

11) C

12) B

13) B

14) D

15) D

16) C

17) A

18) D

19) C

20) B

21) B

22) B

23) D

24) D

25) D

26) D

27) B

28) A

29) A

30) B

31) TRUE

32) TRUE

33) TRUE

34) TRUE

35) FALSE

36) TRUE

37) FALSE

38) FALSE

39) TRUE

40) TRUE

41) TRUE

42) TRUE

43) TRUE

44) TRUE

45) FALSE

  1. very large segments of DNA
  2. Southern blot / Northern blot
  3. synonymous codons
  4. normal restriction and modification systems / recombination pathways
  5. agarose gel / sizes (or lengths)
  6. molecular cloning

mach inery required to splice out introns.

  1. Answers will vary, but a common method is to digest the DNA sample and clone the fragments into a vector. The particular vector will vary depending on the size range of the digested fragments (e.g., cosmids, BACs, HACs, YACs).
  2. The BAC vector itself is quite small (6.7 kbp), containing minimal genes such as oriS and repE for replication in a host as well as sopA and sopB to minimize the copy number. The host strain should also lack modification and restriction systems to prevent the vector from being digested. A recombination defective host is also important to prevent recombination of the host's chromosome with the BAC vector.
  3. A YAC must contain an origin of replication specific to the host, telomeres to ensure the DNA termini are replicated, and centromeres for segregation during mitosis.
  4. Answers will vary, but two possible answers are reporter constructs where reporter proteins are observed (e.g., fluorescence) and blue-white screening of the colonies themselves for disruption/loss of the LacZ reporter.
  5. Answers will vary, but in the example of constructing a library, a restriction enzyme will generate varying sizes with no particular order except the cut site remains the same. Other possible answers should focus on the idea of restriction enzymes cutting at specific sites, which are in some ways randomly dispersed throughout a sequence.
  6. A polylinker, also called a polyclonal site, contains several recognition sequences for different restriction endonucleases to cleave. If a gene is to be ligated into a digested (linearized) vector, the gene itself should also have compatible ends and therefore needs to be digested by the same restriction enzyme. This can be especially problematic if the gene of interest contains a recognition cut site in the middle of its sequence, because then only fragments of the gene could be ligated into the vector. To assuage this issue, a suite of different restriction sites is often housed within a vector.
  7. First, the DNA sequence of interest (gene) must be isolated. The isolated DNA is often larger than the gene of interest, so cutting the short gene sequence out of the large sequence is often performed, which uses restriction enzymes. Inserting the gene into a vector is the second main step. This usually requires DNA ligase whether the insert and vector are blunt- or sticky-ended. The final step involves transforming an (host) organism to house the clone vector.
  8. DNA is negatively charged due to its phosphate backbone, and because opposite charges are attracted to each other DNA migrates towards the positive electrode. A large DNA size (or length) runs slower on a gel, because the agarose (or polyacrylamide) within the gel acts as a physical barrier to impede and slow down the larger DNA molecules.
  9. Artificial chromosome vectors are generally best suited for carrying large inserts of DNA compared to other traditional plasmids. During DNA sequencing of an organism, several overlapping reads are necessary to develop a consensus sequence and therefore mandates a large number of different inserts to be sequenced. With the example of the human genome, an immense number of 1,000 bp fragments would need to be sequenced, all housed within an isolated bacterium containing the plasmid. Larger artificial chromosome decrease the complexity of genome sequencing projects by containing larger inserts (e.g., 800,000 bp fragments) so that fewer individual cells and fewer artificial chromosome vectors need to be sequenced.
  10. All blunt-ended products are capable of joining together, however not all sticky ends will bind to each other. For example, an overhang of one G requires that another product must contain one nucleotide overhang and must be the complementary nucleotide (C). This usually means sticky-ended products must be digested by the same restriction endonuclease.
  11. This is a powerful screening method, because white colonies contain a gene inserted into their plasmid whereas blue colonies do not. In the example of pUC19, the lacZ gene contains a polylinker site where the plasmid can be digested and have a foreign gene

clone d into the site. The

plasmid is re-circularized by DNA ligase such that now the lacZ gene and therefore β-galactosidase (LacZ) activity is disrupted. When transformed cells are grown on a medium with the artificial Xgal substrate, all of those without an insert will have a functional LacZ and will convert the Xgal to a blue color. A white colony suggests the cells contain an inserted gene within the lacZ gene of the plasmid, and these white colonies are likely to be the ones of interest.

  1. Site-directed mutagenesis involves changing individual nucleotides at precise locations within a sequence in vitro. Using short synthetic oligonucleotides (primers) with a mismatched base pair, the mutation site will be amplified with several rounds of PCR. An application to this method is to change nucleotides so that certain amino acids are changed. This circumvents issues associated with codon bias when cloning a gene into a host with a different codon usage pattern. Changing specific nucleotides could also change the recognition site for a restriction endonuclease to either create or remove a specific restriction site. A third application is to introduce several point mutations for the disruption of a gene to study its loss in phenotype and therefore identify its function.
  2. Operon and protein fusions are both examples of gene fusions that bring together two separate nucleotide sequences. A gene fusion is formed when one of the genes' promoter regions is fused to the coding region of the other gene upstream of it. Protein fusions have transcription occur at the same site for the two fused genes, and translation of the fused transcript takes place at one site. An operon fusion retains separate translational start sites despite sharing the same transcriptional start site.