Module 11

Question 1

Gene Cloning

Answer 1

Technique of isolating and making many copies of a gene

Utilized in DNA sequencing, gene editing, DNA probes, and expression of clones

Question 2

Insertion of DNA into Vector

Answer 2

Requires cutting (restriction endonucleases) and joining of DNA fragments

Restriction enzymes can digest DNA into fragments with “sticky ends” that can bind to complementary strand, while others have “blunt ends”

Question 3

Complementary DNA (cDNA)

Answer 3

A DNA strand that does not contain introns and only exons (protein-encoding genes), formed from reverse transcriptase on mRNA

Question 4

DNA Library

Answer 4

Variety of DNA fragments inserted into vectors produces a collection of recombinant vectors

If starting was chromosomal DNA: genomic library
If starting was cDNA: cDNA library

Question 5

Polymerase Chain Reaction (PCR)

Answer 5

Copies DNA without vectors or host cells, requiring template DNA, oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), and Taq polymerase

Question 6

3 Steps of PCR

Answer 6

1. Denaturation: DNA strands are separated
2. Primer Annealing: Oligonucleotide primers bind to DNA strands
3. Primer Extension: Nucleotides are added to primers, extending their lengths

Question 7

Reverse Transcriptase PCR

Answer 7

Used to detect and quantify the amount of RNA in living cells, beginning with RNA isolated from a sample, mixed with reverse transcriptase and a primer which anneals to 3’ end of RNA

Generates ss cDNA which can be used as a template

Question 8

Real-Time PCR

Answer 8

Used to quantitate the amount of specific gene or mRNA in a sample, carried out in a thermocycler that can determine fluorescent changes using the TaqMan system

Question 9

TaqMan System

Answer 9

Detects oligonucleotide with a fluorescent reporter molecule and a quencher molecule at either end. As the replicated length increases the fluorescence gets brighter, eventually reaching the cycle threshold

Question 10

Cycle Threshold (C_t)

Answer 10

The point at which the accumulation of fluorescence in real-time PCR is significantly greater than the background level

Question 11

Dideoxy DNA Sequencing

Answer 11

Based on DNA replication, a single tube contains all 4 nucleotides along with 4 fluorescently tagged ddNTPs without 3’ OH groups

Sanger sample is loaded onto single gel lane after polymerization, color fluoresces at each location identifying the specific nucleotide at that spot in the sequence

Question 12

Gene Editing

Answer 12

Altering the sequence of a gene experimentally

Question 13

CRISPR-Cas Editing Technology

Answer 13

Creates single guide RNA complex made of tracrRNA and crRNA. sgRNA then guides Cas9 to gene sequence using its spacer region which is complementary to the target gene

Question 14

Cas9 cut can be repaired either through ____ which deactivates gene, or through ____ which allows for the insertion of donor DNA (causing point mutation)

Answer 14

non homologous end joining (NHEJ), homologous recombination repair (HRR)

Question 15

Northern Blotting

Answer 15

Used to identify a specific RNA in a mixture to determine if a gene is transcribed within a cell type, at a development stage, and also observe if the pre-mRNA is alternatively spliced

Extract RNA mixture, separate via gel electrophoresis, blot onto nitrocellulose fiber, place in solution containing a probe, complementary DNA appears as a dark band

Question 16

Western Blotting

Answer 16

Used to identify a specific protein in a mixture to determine if a protein is made within a cell type or at a development stage

Protein mixture separated via SDS-PAGE, denaturing them and coating with negative charges. Protein bands in gel are blotted with nitrocellulose which is placed in solution of primary and secondary (conjugated to alkaline phosphatase) Abs, protein of interest revealed as a dark band

Question 17

Microorganisms in biotechnology are used for:

Answer 17

medicine, agriculture, biological control, and bioremediation

Question 18

Insulin from Bacteria

Answer 18

Recombinant bacteria form the A and B chains of insulin separately, with each chain stabilized by a methionine and beta-galactosidase which are cleaved allowing for A and B to join

Question 19

Biological Control

Answer 19

Use of microorganisms or products to alleviate plant problems (e.g. disease from environmental conditions)

Question 20

Bioremediation

Answer 20

Use of microorganisms to reduce environmental pollutants by transforming structure of toxic pollutant (biotransformation) resulting in biodegradation

Question 21

Transgenic Organism

Answer 21

An organism containing genes from other individuals/species

Question 22

Gene Knock-in Mice

Answer 22

Utilizes CRISPR-Cas technology and homologous recombination repair to insert a gene from another organism into the mouse genome at a noncritical site in an oocyte

Question 23

Molecular Pharming

Answer 23

Creation of transgenic livestock which are encoded to produce important human proteins in their mammary glands

Question 24

Dolly the Sheep

Answer 24

1. A diploid somatic mammary cell from a donor sheep was combined with an unfertilized egg
2. The egg nucleus was removed and the cells were fused with an electrical pulse, initiating the development of a zygote
3. The embryo was transferred into a surrogate ewe
4. Dolly is born! Genetically identical to the donor sheep

Question 25

Stem Cells

Answer 25

Supply cells that construct bodies from a fertilized egg Can replenish damaged cells in adults and vary in their capacity to proliferate and differentiate into one or more specialized cell types

Question 26

Medical Interest in Stem Cells

Answer 26

They allow us to understand the basic genetic mechanisms that underlie development and allow for the treatment of human disease and injury