DNA Manipulation: Enzymes, CRISPR, PCR, and GMOs

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Questions and Answers

Which enzyme is used to catalyze the formation of a phosphodiester bond between two DNA fragments?

  • Polymerase
  • Reverse transcriptase
  • Endonuclease
  • Ligase (correct)

A scientist wants to amplify a specific segment of DNA. Which technique should they use?

  • Gel electrophoresis
  • Southern blotting
  • Polymerase Chain Reaction (PCR) (correct)
  • Restriction digestion

Which of the following accurately describes the function of endonucleases?

  • Synthesizing new DNA strands.
  • Amplifying segments of DNA.
  • Cutting DNA at specific sequences. (correct)
  • Joining DNA fragments together.

After cutting a DNA sequence with a specific restriction enzyme, a researcher notices the DNA fragments have overhanging ends. What are these ends called?

<p>Sticky ends (A)</p> Signup and view all the answers

Which of the following is true regarding blunt end ligation?

<p>DNA fragments are joined directly together. (D)</p> Signup and view all the answers

What role does reverse transcriptase play in molecular biology?

<p>Synthesizing DNA from an RNA template. (A)</p> Signup and view all the answers

In gel electrophoresis, what property of DNA allows it to be separated?

<p>Molecular weight (B)</p> Signup and view all the answers

During electrophoresis, what causes the separated DNA fragments to move through the gel?

<p>An electric field (D)</p> Signup and view all the answers

What is ethidium bromide used for in gel electrophoresis?

<p>To make the DNA visible under UV light. (A)</p> Signup and view all the answers

What is the term for a segment of DNA used to locate a specific, complementary sequence?

<p>Probe (B)</p> Signup and view all the answers

What is the primary difference between nuclear DNA and mitochondrial DNA used for identification?

<p>mtDNA is almost exclusively inherited via the maternal line, nuclear DNA is inherited from both parents. (B)</p> Signup and view all the answers

What are Short Tandem Repeats (STRs)?

<p>Long sequences of non-coding Introns (D)</p> Signup and view all the answers

What is required for a individual to be defined as a GMO?

<p>Any gene or DNA segment that is added through genetic engineering should be heritable (D)</p> Signup and view all the answers

Herbicide-resistant crops were first altered to be uneffected by which herbicide?

<p>Glyphosate (C)</p> Signup and view all the answers

Which of the following crops produces a natural pesticide?

<p>Cotton (B)</p> Signup and view all the answers

Which of the following explains Gene Cloning?

<p>Locating a gene of interest to to produce multiple copies (D)</p> Signup and view all the answers

What describes a recombinant plasmid?

<p>A plasmid that carries foreign DNA. (A)</p> Signup and view all the answers

What main 2 factors are used to form recombinant Plasmids?

<p>Endonucleases with with ligase (A)</p> Signup and view all the answers

What does bacterial transformation achieve?

<p>Transferring recombinant plasmids into bacterial cells. (D)</p> Signup and view all the answers

One way recombinant plasmids into bacterial cells is low, bacteria can be increased through several steps, which of the following is not one of those steps?

<p>Lysosomes (A)</p> Signup and view all the answers

What is indicated when bacterial colonies appear white when grown on X-gal after transformation?

<p>They contain the gene of interest within the plasmid (C)</p> Signup and view all the answers

Which of the following is an example of a recombinant protein that can be used to to produce human insluin?

<p>proinsulin (B)</p> Signup and view all the answers

Which phrase describes the end result of Electroporation?

<p>Cells are briefly placed in an electric field. (B)</p> Signup and view all the answers

The development of GMOs may involve the silencing of a gene, what does that achieve?

<p>so that its function is lost. (C)</p> Signup and view all the answers

When an engineer is identifying a transgenic organism, that determines that it is transgenic?

<p>The proteins has origin from a different species.. (C)</p> Signup and view all the answers

Golden Rice is known for increased levels of which important vitamin?

<p>beta-carotene (C)</p> Signup and view all the answers

Which vector is responsible used to ingeneer the gox gene in canola plants to create Resistant Roundup canola??

<p>Agrobacterium (B)</p> Signup and view all the answers

What type of insecticide is the cry1Ac gene?

<p>Protein-based (C)</p> Signup and view all the answers

CRISPR uses what to precisely change part of a genome by completing actions such as removing or editing sections?

<p>Single Guide RNA (C)</p> Signup and view all the answers

What did Barrangou and his team identify as the role of CRISPR-Cas9 in bacteria?

<p>Adaptive immunity (B)</p> Signup and view all the answers

Which component of the CRISPR-Cas9 system acts as a guide, directing the complex to the specific target DNA sequence?

<p>The guide RNA (sgRNA) (C)</p> Signup and view all the answers

What is the natural function of CRISPR-Cas9 system in bacteria?

<p>Adaptive Immunity (B)</p> Signup and view all the answers

Before the use of CRSPR or more modern methods, what did early genetic manipulations use to inject cells with?

<p>Modified retroviruses (D)</p> Signup and view all the answers

In the CRISPR-Cas9 system, what role does the PAM (protospacer adjacent motif) sequence play?

<p>Serves as a tag to signal cut recognition (D)</p> Signup and view all the answers

Why is it important to consider whether DNA is circular or linear when determining the number of fragments produced after restriction enzyme digestion?

<p>The number of fragments produced differs based on topology (B)</p> Signup and view all the answers

What feature of bacterial cells is most critical to their use in recombinant DNA technology for producing human proteins?

<p>Introns (A)</p> Signup and view all the answers

In which of the following scenarios would mtDNA analysis be most advantageous over nuclear DNA analysis?

<p>Identifying the remains of a soldier (C)</p> Signup and view all the answers

Flashcards

Polymerases

Enzymes that synthesise DNA.

Ligases

Enzymes that join DNA fragments.

Endonucleases

Enzymes that cut DNA at specific sites.

CRISPR-Cas9

A tool for precise genome editing using RNA to guide an endonuclease to cut DNA.

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PCR

A technique to amplify a segment of DNA.

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Reverse transcriptase

Enzyme that directs copy DNA formation from mRNA.

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cDNA

DNA strand with bases complementary to the opposite strand, produced via reverse transcription.

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Restriction site

The recognition sequence of a restriction enzyme.

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Blunt ends

Ends of a DNA fragment with no overhanging bases after being cut by an endonuclease.

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Sticky ends

Ends of a DNA fragment having overhanging bases after being by an endonuclease

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Gel electrophoresis

A technique for sorting DNA fragments through an electric field.

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DNA fingerprinting

Technique to identify DNA from individuals based on short tandem repeat variations.

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Endonucleases

Enzymes that cuts at specific sites within DNA. Also known as restriction enzymes.

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DNA sequencing

Identifying base sequence along a DNA strand.

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Hypervariable regions

Regions in DNA that are highly polymorphic.

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Short tandem repeats (STRs)

Chromosomal sites where many copies of a short DNA sequence are joined end-to-end.

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Genome editing

Process by which changes are made to the nucleic acid sequence of genes

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Plasmid

Small circular DNA able to reproduce independantly that may be taken up by cells.

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Antibiotic resistance gene

Marker genes governing antibiotic resistance.

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Origin of replication (ORI)

Section of DNA sequence which is recognised by a cell's DNA replication proteins

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Recombinant plasmids

Plasmids that carry foreign DNA.

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Vector

An agent or vehicle used to transfer pathogens/genes.

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Bacterial transformation

A process where bacterial cells take up segments of foreign DNA that become part of their genetic make-up

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Electroporation

A technique that uses brief exposure of host cells to an electric field to enable the entry of foreign DNA into the cells

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Heat shock

A technique to transform bacteria in which cells are suspended in a ice cold solution and then moved into a warm to increase plasma membrane fluidity

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Genetically modified organisms (GMOs)

Those organisms whose genomes has been altered through genetic engineering technology

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Transgenic organisms

Organism that carry, in their genomes, one or more genes artificially introduced from another species.

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Study Notes

  • Topic 2 covers DNA manipulation techniques and applications, focusing on enzymes used for manipulation, CRISPR-Cas9, PCR, gel electrophoresis, recombinant plasmids, and genetically modified organisms

DNA Manipulation Techniques

  • Enzymes consist of polymerase, ligase, and endonucleases, each used to synthesize, join and cut DNA respectively
  • CRISPR-Cas9 is a bacterial immune system, adapted to editing an organism's genome by cutting and modifying DNA sequences at precise locations

PCR, Electrophoresis, and Plasmids

  • PCR amplifies DNA segments, enabling large quantities of specific sequences to be obtained from very small samples
  • Gel electrophoresis sorts DNA fragments by size, primarily used in DNA profiling and forensics
  • Recombinant plasmids are vectors used to to transform bacterial cells

Enzymes Manipulating DNA

  • Genetic engineers mostly work with genetic material, especially DNA, therefore, the genetic engineer requires tools to cut, join, copy, and separate DNA.
  • DNA polymerase was identified by Arthur Kornberg in 1956.

Synthesis of DNA

  • If specific genes or sections of DNA are needed, using mRNA as a template is useful
  • Reverse transcriptase uses mRNA to build single-stranded DNA with a complementary base sequence
  • Second strand created with polymerase
  • This DNA is known as cDNA
  • Reverse transcriptase has copied the human growth hormone gene

The role of endonucleases

  • Endonucleases (restriction enzymes) cleave DNA at restriction sites, with each enzyme recognizing different sequences
  • Enzymes can cut at points directly opposite each other, then use blunt ends to ligate
  • Other endonucleases cut one strand at one point, then cut the second strand at a point slightly away
  • This results in sticky ends, allowing scientists more control over where ligation occurs

Restriction Cutting

  • A DNA sample is dissolved then the particular endonuclease is added to it
  • The result is the DNA sample will be cut into 2 or more fragments

DNA profiling

  • DNA profiling identifies individuals using a technique because each person has unique regions of DNA
  • DNA can be amplified through PCR to carry out DNA profiling
  • A constant DNA profile enables identification
  • DNA can be taken from any cell, blood, somatic cell, etc
  • An example of DNA profiling includes comparing one to the national DNA database

Gene editing through CRISPR-Cas9

  • CRISPR-Cas9 involves cutting DNA and using a guide to direct the scissors to a site in the DNA
  • The guide RNA directs the cellular machinery to a defined genomic target
  • Editing means correcting, adding/replacing text, or deleting a sentence

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