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3) Genetics > Chapter 14: Molecular Genetic Analysis and Biotechnology > Flashcards

Chapter 14: Molecular Genetic Analysis and Biotechnology Flashcards

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1
Q

Genetically engineered corn, which produces a toxin that kills insect pests, constitutes what percentage of all corn grown in the United States?

A

85%

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2
Q

What sequences are required for the transfer of the DNA within the TI plasmid from the bacteria to the plant genome?

A
  • Flanking sequences TL and TR

- The DNA between TL and TR integrates into the genome of the plant cell

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3
Q

What plasmid may be used to transfer genes to plants?

A

The TI plasmid

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4
Q

What do the elements contained within the DNA of the Ti plasmid allow for?

A

Their proliferation following their integration into the plant cell

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5
Q

Describe how a foreign DNA may be integrated into a plant chromosome.

A

1) Foreign DNA is inserted into a plasmid vector and transferred to a bacterium with the plasmid
2) The Ti plasmid vector is transferred to a plant cell, where it integrates into a plant chromosome

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6
Q

What is required for the differentiation between plasmids?

A

Selectable marker

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7
Q

What must be used for transformation to create a stable line?

A

A germline cell must be transformed to create a stable transgenic line

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8
Q

What are the two ways to transform a germline cell to create a stable transgenic line?

A

1) Arabidopsis: make the plant flower and dip it into the bacteria
2) Maize: grow on plant, take immature seed, and remove the embryo, place on petri dish to grow, and put into contact with the bacterium

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9
Q

What are the downsides of transgenes?

A
  • Requires selectable markers, which are pieces of foreign DNA; their transfer carries a risk (ex: transferring antibiotic resistance)
  • Crossing the species barrier (ex: create a plant with a human gene)
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10
Q

What was the Bt toxin gene used for? What does it encode for?

A
  • Encodes for an insecticide

- Isolated from bacteria and transferred to tobacco plants

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11
Q

What is the CRISPR system essentially?

A

The immune system of a bacteria

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12
Q

What is the function of the Cas9 enzyme?

A
  • Interacts with a piece of RNA (sgRNA), which matches a gene that is unwanted within the system
  • Cas9 cuts the sequence, and usually creates a frameshift that destroys the gene
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13
Q

How can we use the CRISPR/Cas9 system to our advantage? What must occur afterwards?

A
  • If we create a guide RNA (sgRNA), the Cas9 system may be told to travel and cut wherever we want
  • The creation of a transgene occurs, so the Cas9 gene must be crossed-out
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14
Q

What is the function of the polymerase chain reaction?

A

Amplification of short DNA fragments, even if they are present in small amounts

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15
Q

What enzyme is used in PCR? Why?

A
  • Taq polymerase

- Remarkably stable at high temperatures, and is not denatured in the strand separation step of PCR

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16
Q

Describe the PCR process.

A

1) 95oC: DNA strands are separated
2) 45oC: primer appealing
3) 72oC: DNA polymerase synthesizes new strands

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17
Q

What is present in the solution for PCR?

A
  • Target DNA
  • DNA polymerase
  • All four dNTPs
  • Primers (forward and reverse)
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18
Q

What are restriction enzymes?

A

Enzymes that recognize and make double-stranded cuts in DNA at specific nucleotide sequences

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19
Q

What are cohesive ends?

A
  • Also called sticky ends
  • Complementary to each other and can spontaneously pair to connect the fragments
  • Any two fragments cleaved by the same enzyme will have complementary ends and will pair
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20
Q

What are the two types of ends produced by restriction enzymes?

A
  • Blunt ends

- Cohesive ends (sticky ends)

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21
Q

What are restriction fragment length polymorphisms used for?

A
  • Genetic markers that can be used in mapping
  • Certain bands produced are distinct to a specific individual
  • However, a significant amount of DNA is required for this technique
22
Q

What does an idealized cloning vector contain?

A
  • An origin of replication
  • One or more selectable markers
  • One or more unique restriction sites (multi-cloning sites)
23
Q

Where do restriction enzymes come from?

A

Exist naturally in bacteria, which use them in defense against viruses

24
Q

What is gene cloning?

A

The amplification of a specific piece of DNA by placing the fragment in a bacterial cell, allowing the cell to replicate the DNA

25
Q

What is a cloning vector?

A

Stable, replicating DNA molecule into which a foreign DNA molecule can be inserted for introduction into a cell

26
Q

What does the pUC19 plasmid contain?

A
  • Cluster of unique restriction sites
  • Origin of replication
  • Ampicillin-resistance gene (selectable marker)
  • LacZ gene (selectable marker)
27
Q

What may the LacZ gene be used to screen?

A

Screen bacteria containing recombinant plasmids

28
Q

What is the easiest method for inserting a particular DNA sequence into a plasmid vector?

A

Cut the foreign DNA and the plasmid with the same restriction enzyme, and then ligate

29
Q 
DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length are separated by gel electrophoresis. Which fragment will migrate farthest in the gel?
A) The 2000-bp fragment
B) The 1000-bp fragment
C) The 500-bp fragment
D) All will migrate equal distances
A

C) The 500-bp fragment

30
Q

What occurs in DNA gel electrophoresis? What does it allow?

A
  • Negatively charged DNA migrates towards the positive side through a gel
  • Separates DNA molecules on the basis of their size and charge
31
Q

What is a probe?

A
  • DNA or RNA molecule with a base sequence complementary to a sequence in the gene of interest
  • The probe only pairs with the bases on the complementary sequence, so it can locate a specific gene
32
Q

Describe Southern blotting.

A

1) DNA is cut into fragments using restriction enzymes
2) Separates the fragments on a gel
3) Fragments are denatured and transferred to a membrane
4) Membrane is placed in a hybridization solution with a labeled probe
5) Biochemical method reveals the presence of the bound probe

33
Q

What does Southern blotting allow?

A

Can reveal the presence of a specific DNA fragment in a genome or sample of DNA

34
Q

What is Northern blotting?

A

RNA is transferred from a gel to a solid medium

35
Q

What is Western blotting?

A
  • Transfer of protein from a gel to a membrane

- The probe is usually an antibody

36
Q

What is a genomic library?

A

Contains all of the DNA sequences found in an organism’s genome

37
Q

How may DNA libraries be screened (positional cloning)?

A
  • Fragments of DNA are joined to a cloning vector and placed on a Petri dish
  • Imprint of the colony is copied, and put in contact with a probe to reveal which colonies contain the fragment of interest
  • Comparison of the membrane with the master plate reveals which bacterial colonies have the DNA of interest
38
Q

What is a cDNA library?

A

Contains only DNA sequences that are transcribed into mRNA

39
Q

What is positional cloning?

A
  • Isolation of genes on the basis of their position on a gene map
  • After the locus has been mapped, clones that cover the region can be isolated, and all genes within the region can be identified
40
Q

What are restriction fragment length polymorphisms? What may they be used for?

A
  • Variations in the patterns of fragments produced when DNA molecules are cut with the same restriction enzyme
  • Genetic markers that can be used in mapping, as these differences are inherited
41
Q

What is the function of DNA sequencing?

A

Reveals the nucleotide base sequence in DNA

42
Q

What is the substrate utilized in the dideoxy-sequencing reaction?

A
  • Dideoxynucleotides (ddNTPs)
  • Contain an H at the 3’ carbon instead of an OH
  • The OH group is used to attach subsequent nucleotides
  • The removal of OH allows ddNTPs to terminate DNA synthesis
43
Q

How does the incorporation of ddNTPs into the new strand allow for the sequencing of DNA?

A
  • Incorporation takes place randomly at different positions in different copies, producing a set of DNA fragments of different lengths
  • Fragments are separated by gel electrophoresis
  • Sequence can be read from the bands on the gel, starting at the bottom
44
Q

In the dideoxy sequencing reaction, what terminates DNA synthesis at a particular base?
A) The absence of a base on the ddNTP halts the DNA polymerase
B) The ddNTP causes a break in the sugar-phosphate backbone
C) DNA polymerase will not incorporate a ddNTP into the growing DNA strand
D) The absence of a 3’-OH group on the ddNTP prevents the addition of another nucleotide

A

D) The absence of a 3’-OH group on the ddNTP prevents the addition of another nucleotide

45
Q

What makes Next Generation Sequencing Technologies sequence genomes faster?

A

Sequencing in parallel, which means that millions of DNA fragments can be sequenced simultaneously

46
Q

What is DNA fingerprinting?

A

The use of DNA sequences to identify individual persons

47
Q

What does most DNA fingerprinting use?

A

Microsatellites

48
Q

What are microsatellites?

A
  • Short DNA sequences repeated in tandem, which are found at many loci throughout the human genome
  • People vary in the number of copies of repeat sequences they possess at each of these loci
49
Q

How are microsatellites detected?

A

Detected by using PCR with primers that flank the region containing the repeats

50
Q

How is a gene inserted into a plasmid cloning vector?

A
  • The gene and plasmid are cut with the same restriction enzyme and mixed together.
  • DNA ligase is used to seal nicks in the sugar–phosphate backbone.

3) Genetics (16 decks)

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