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3: Recombinant DNA Technology Flashcards

1
Q

4 Applications of Recombinant DNA Technology

A
  1. Transgenic animals
  2. Nucleic Acids
  3. Transgenic plants
  4. Recombinant Microbes
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2
Q

Recombinant DNA technology involves using _ and _ to manipulate and isolate DNA segments of interest.

A

enzymes
various laboratory techniques

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3
Q

cutting and pasting DNA from different
samples/specimen

A

Recombinant DNA Technology

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4
Q

_ often relies on recombinant DNA technology and gene cloning to modify an organism’s genome

A

genetic engineering

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5
Q

3 Classifications of Enzymes in Recombinant DNA Technology

A

Nucleases
DNA modifiers
DNA ligase

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6
Q

3 Nucleases

A
  1. Restriction endonucleases
  2. restriction exonucleases
  3. ribonucleases H
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7
Q

6 DNA modifiers enzyme

A
  1. DNA polymerase
  2. Reverse transcriptase
  3. alkaline phosphatase
  4. polynucleotide kinase
  5. terminal nucleotidyl transferse
  6. methyl transferase
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8
Q

enzymes that joins the DNA fragments with
cloning vector

A

DNA ligase

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9
Q

3 Steps of DNA Ligation

A
  1. DNA ligase is self-adenylated
  2. Adenyl group is transferred
  3. Phospodiester bond forms
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10
Q

enzyme that synthesize complementary
strand (cDNA) from mRNA template

A

reverse transcriptase/
RNA dependent DNA polymerase

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11
Q

3 Functions of reverse transcriptase/
RNA dependent DNA polymerase

A
  1. synthesize cDNA
  2. amplify cDNA
  3. analyze mRNA
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12
Q

enzyme that recognize and cut DNA strand at specific sequence called restriction site

A

restriction endonuclease

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13
Q

3 types of restriction endonucleases

A

Type 1 RE
Type II RE
Type III RE

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14
Q

3 types of restriction endonucleases

recognize a bipartite sequence but do not produce a predictable cleavage pattern

A

Type 1 RE

1 enzyme w 3 subunits for recognition cleavage and methylation

1000bp from recognition site
E. coli
Citrobacter fruendii

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15
Q

3 types of restriction endonucleases

most commonly used for molecular biology applications as they reconize stereotypical sequences and produce a predictable cleavage pattern

A

Type II RE

2 diff enzymes; same as recognition/ close to recognition site

Escherichia coli
Arthrobacter luteus

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16
Q

3 types of restriction endonucleases

recognize a non-palindromic sequence, comprising two inversely oriented sites

A

Type III RE

1 enzyme with 2 subunits; 24-26 bp from recognition site

Haemophilus influenzae
Providencia stuarti

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17
Q

Enzymes used in recombinant DNA technology

are enzymes composed of distinct domains that exhibit different biochemical activities.

It converts blunt end of DNA fragments into sticky end.

A

terminal transcriptase

Sticky ends are helpful in cloning because they hold two
pieces of DNA together so they can be linked by
DNA ligase

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18
Q

Enzymes used in recombinant DNA technology

It usually cut DNA on either side of distortion caused by thymine dimers or intercalating agents.

A

Nuclease

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19
Q

Enzymes used in recombinant DNA technology

synthesize nucleotide complementary to template strand and helps to fill gap in double stranded DNA.

A

DNA polymerase

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20
Q

Enzymes used in recombinant DNA technology

removes mRNA from DNA-RNA heteroduplex and that mRNA is used to synthesize cDNA

A

Ribonuclease-H

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21
Q

Enzymes used in recombinant DNA technology

helps in
removal of terminal phosphate group
from 5′ end

A

alkaline phosphatase

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22
Q

Enzymes used in recombinant DNA technology

It adds phosphate group from ATP molecule to
terminal 5’end after dephosphorylation
by alkaline phosphatase

A

Polynucleotide kinase

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23
Q

Enzymes used in recombinant DNA technology

enzymes that transfer a methyl group from S-denosylmethionine (SAM) to their substrates.
DNA methyltransferases modify DNA by
adding a methyl group to cytosines.

A

Methyltransferases

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24
Q

Type of Nuclease

hydrolyzing enzyme that
cleaves the phosphodiester bond between the nucleotides

A

endonuclease

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25
Q

Type of Nuclease

cleave from the ends

A

exonuclease

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26
Q

Clone is derived from Greek word that describes _ that used to propagate or copy a plant

A

cutting (of a twig)

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27
Q

a molecule, cell, or organism produced
from another single entity to which it shares
the same genetic make-up.

A

clone

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28
Q

the process of producing or
generating a genetically identical
copy of a cell or an organism.

A

cloning

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29
Q

DNA cutting enzymes, also known as
“scissors” used for gene cloning

A

Restriction enzymes

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30
Q

Restriction enzymes
aka _ because they cut within DNA sequences as
opposed to enzymes that cut from the ends of
DNA sequences (exonucleases)

A

restriction endonucleases

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31
Q

are the DNA molecules, which can carry a foreign DNA fragment into a host cell

self replicating

A

vectors

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32
Q

it is used for the reproducing the DNA fragment

A

cloning vector

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33
Q

vectors aka _ because they act as carrier of gene to be cloned into a recipient cell

A

vehicle DNA

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34
Q

properties of vector

A
  1. small DNA molecules
  2. origin of replication
  3. unique restriction enzyme are able to replicate autonomously
  4. be non-toxic to host cell
  5. have space for foreign insert
  6. the vector should have suitable marker genes that allow easy selection of the transformed host cell
  7. unique recognition site
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35
Q

are DNA molecules into which foreign DNA can be inserted

A

cloning vectors

36
Q

vectors are also known as _ for delivering foreign DNA into recipient cells

37
Q

during _ , the desired DNA is carried by a vector into another cell where it can be replicated and/or expressed

A

DNA cloning

38
Q

vector is an artifically synthesized manipulated DNA whereas a _ naturally occurs in bacterial cells

39
Q

there are several _ which can be used in recombinant DNA, whereas all _ may not be used directly in recombinant DNA technology

A

vectors
plasmids

40
Q

Preparation of recombinant plasmid vector

A
  1. cleave vector with restriction enzyme
  2. cleave foreign DNA with same restriction enzyme
  3. mix vector and DNA fragment under conditions that favor base pairing
  4. treat with DNA ligase to join DNA pieces covalently
41
Q

the sequence at which replication of DNA begins

A

ori
origin of replication

42
Q

Ori is also linked to the copy _ and so controls how many times the gene of interest will be produced in the host cell

A

number of vector

43
Q

_ are genes that help identify bacteria that have successfully transformed, or taken up the recombinant plasmid

_ are commonly used markers

A

selectable markers

antibiotic resistance genes

ideally, the enzyme should have 1 or max of 2 recognition sites

44
Q

why should the number of enzymes be so low

A

cutting the vector within the selectable marker will interrupt that genes, and allow for identification of recombinants

ex. using cloning site within an ampicillin resistance gene will result in recombinants that are no longer resistance to that antibiotic

45
Q

_ region of the gene acts as a light switch

signals when to turn the gene on and off

46
Q

a short region of DNA (100-1000 bp) where transcription of a gene by RNA polymerase begins

47
Q

promoter is typically located directly_ or at the _ end of the transcription initiation site

A

upstream
5’

48
Q

Characteristics of cloning vectors

  1. _ inside host cell
  2. possess a _ for RE enzymes
  3. _ by the introduction of donor DNA fragment in terms of its replication property
  4. possess some _ such that it can be used for identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell)
  5. _ from host cell
A
  1. self-replicating
  2. unique restriction site
  3. unaffected
  4. marker gene
  5. early isolated
49
Q

Types of Cloning vectors

A
  1. bacteriophage
  2. plasmid DNA/ plasmid vector
  3. bacterial artificial chromosomes BACs
  4. yeast artificial chromosomes YACs
  5. human artificial chromosomes HACs
50
Q

Types of Cloning Vectors

are viruses which can infect bacterial cells

capable to deliver DNA fragment of a size up to 20kb

A

bacteriophage

51
Q

Types of Cloning Vectors

small circular pieces of DNA found in primarily in bacteria

considered as extrachromosomal DNA in bacteria (found in the cytoplasm in addition to the bacterial chromosome)

A

plasmid DNA
plasmid vector

52
Q

plasmids may be inserted into bacterial cells in the process known as

a DNA fragment of size up to 10kb can be delivered using this vector

A

transformation

53
Q

Types of Cloning Vectors

plasmid which is designed to clone
very large DNA fragments ranging in
size from 75 to 300 kb

A

Bacterial Artificial Chromosomes
(BACs)

54
Q

Bacterial Artificial Chromosomes
(BACs) used in sequencing the genome of
organisms in genome projects i.e., _

A

human genome project

55
Q

Types of Cloning Vectors

  • _ expression vectors
  • may clone DNA fragments with sizes from 100
    kb to 3000 kb
  • used for cloning very large DNA fragments and
    for the physical mapping of complex genomes
  • _ have an advantage over BACs in
    expressing eukaryotic proteins that require
    post translational modifications
A

Yeast Artificial Chromosomes (YACs)

56
Q

YACs are known to produce _ which make them less stable compared
to BACs

A

chimeric
effects

57
Q

An organism or tissue that contains at least two different sets of DNA, most often originating from the fusion of as many different zygotes (fertilized eggs).

58
Q

“chimeric effects” : artifacts where the
sequence of the cloned DNA actually
corresponds not to a single genomic region
but to _

A

multiple regions.

Chimeric DNA refers to a DNA fragment that
consists of DNA from two or more different
sources

59
Q

Type of Cloning Vector

  • also known as mammalian artificial chromosomes (MACs)
  • still under development
  • micro-chromosomes that can act as a new chromosome in a population of human cells
  • range in size from 6 to 10 Mb that carry
    new genes introduced by human researchers
A

Human Artificial Chromosomes (HACs)

60
Q

used as vectors in transfer of new genes,
studying their expression, and mammalian
chromosomal function can also be elucidated
using these micro chromosomes in
mammalian system

A

Human Artificial Chromosomes (HACs)

61
Q

DNA fragment/s from
two different species
that are inserted into a
host organisms to
produce new genetic
combinations that could
add value to science,
agriculture, medicine,
and industry.

A

Recombinant DNA

62
Q

2 process of
Identifying and Cloning the Gene of
Interest

A
  1. Shotgun cloning/Shotgun method/Shotgun approach
  2. Cloning approach involving DNA libraries
    2.1 DNA Library
    2.1.1 Complementary DNA
    2.1.2 Genomic Library
    2.2 Library Screening
    2.3 Polymerase Chain Reaction
63
Q

Identifying and Cloning the Gene of Interest

many fragments are randomly cloned at
once and no individual gene is specifically
targeted for cloning.

A

Shotgun cloning/Shotgun method/Shotgun
approach

64
Q

Identifying and Cloning the Gene of Interest

a method for the identification and cloning of
genes which includes a DNA library

A

Cloning approach involving DNA libraries

65
Q

Laboratory Techniques & Application of Recombinant DNA Technology

designed to produce highly accurate and
long stretches of DNA sequence, greater than
1 giga base (billion bases) of DNA per reaction,
at a low cost

A

Next Generation Sequencing (NGS)

66
Q

Laboratory Techniques & Application of Recombinant DNA Technology

  • can be used to identify which chromosome
    contains a gene of interest
  • can also be used to determine the cell type that
    is expressing a particular mRNA
A

Fluorescence In Situ Hybridization (FISH)

67
Q

Laboratory Techniques & Application of Recombinant DNA Technology

  • molecular biology technique for identification
    and quantification of DNA
  • begins by digesting chromosomal DNA into
    small fragments with restriction enzymes
  • following electrophoresis, the gel is treated
    with an alkaline solution to denature the DNA;
    then the fragments are transferred onto a
    nylon or nitrocellulose membrane using a
    technique called blotting
  • used to visualize only specific fragments
    of interest
A

Southern Blotting/Southern blot
analysis/Southern hybridization

68
Q

Laboratory Techniques & Application of Recombinant DNA Technology

  • molecular biology technique for identification and quantification of RNA
  • RNA is isolated from a tissue of interest and
    separated by gel electrophoresis (the RNA is
    not digested with enzymes)
  • RNA is blotted onto a nylon membrane and
    then hybridized to a probe - Amounts of mRNA
    produced by different tissues can be compared
    and quantified via different techniques
A

Northern Blot Analysis

69
Q

collection of cloned
DNA fragments from a particular
organism contained within bacteria or
virus as a host.

A

DNA Library

70
Q

types of libraries for cloning

A

genomic DNA libraries and
complementary DNA libraries (cDNA libraries)

71
Q

is synthetic DNA
that has been transcribed from a specific mRNA
through a reaction using the enzyme reverse
transcriptase.

A

complementary DNA (cDNA)

72
Q

what library talks about the chromosomal DNA (which the whole
genome of the organism) from the tissue of
interest is isolated and then digested with
restriction enzyme .

A

Genomic Library

73
Q

Disadvantage of Genomic Library

A

(1) non-protein-coding pieces of DNA, called
introns, are cloned in addition to protein-coding sequences (exons); and
(2) eukaryotes have large genomes

74
Q

what library talks about the mRNA from the tissue of interest is
isolated and used for making the library

A

cDNA library

75
Q

mRNA cannot be cut directly with
restriction enzymes so it has to be
converted to a _

A

double stranded DNA
molecule (complementary DNA or cDNA)

76
Q

– after building the genomic library or cDNA library, it must undergo _ to identify the genes of interest
- colony hybridization

A

Library Screening

77
Q

a more rapid approach to cloning
compared to building and screening a
library - a technique for making copies or
amplifying a specific sequence of DNA in
a short period of time.

A

Polymerase Chain Reaction (PCR)

78
Q

Some Laboratory Techniques and Application
of Recombinant DNA Technology

A
  1. Agarose gel electrophoresis
  2. DNA sequencing
  3. Next Generation Sequencing (NGS)
  4. Fluorescence In Situ Hybridization (FISH)
  5. Southern Blotting/Southern blot analysis/Southern hybridization
  6. Northern Blot Analysis
79
Q

Laboratory Techniques & Application of Recombinant DNA Technology

  • allows one to separate and visualize DNA
    fragments based on size
  • applications generally involve gels that contain
    0.5% to 2% agarose
A

Agarose gel electrophoresis

80
Q

Laboratory Techniques & Application of Recombinant DNA Technology

a technique of **determining the nucleotide sequence of the gene **– the exact order of the bases in the
genome or gene of the organism

A

DNA Sequencing

81
Q

gel with a high percentage of agarose (say 2%)
is better suited for separating _ DNA
fragments while lower percentage of agarose is
better suited for resolving _ DNA fragments.

A

small

large

82
Q

Benefits of knowing the exact sequence of
the gene

A
  1. to deduce the amino acid sequence of a protein encoded by a cloned gene
  2. to determine the exact structure of gene
  3. to identify regulatory elements such as promoter sequences
  4. to identify differences in genes created by gene splicing
  5. to identify genetic mutations
83
Q

performed when only very small amount of
mRNA is extracted from the sample; isolated
mRNA is converted into double-stranded
cDNA by the enzyme reverse transcriptase in a
process similar to the way in which cDNA for a
library is made

A

Reverse transcription PCR (RT-PCR)

84
Q

enables researchers to quantify amplification
reactions as they occur in “real time”; basic
procedure involves the use of specialized
thermal cyclers that use a laser to scan a beam
of light through the top or bottom of each PCR
tube

A

Real-time PCR or quantitative PCR (qPCR)

85
Q

another technique for studying gene
expression; created with the use of a small
glass microscope slide; single-stranded DNA
molecules are attached or “spotted” onto the
slide using a computer-controlled high-speed robotic arm called an arrayer, which
is fitted with a number of tiny pins; arrayer
fixes the DNA onto the slide at specific
locations

A

Gene microarrays/DNA microarrays/gene
chip

86
Q

mutations can be
created in specific nucleotides of a cloned gene
contained in a vector; gene can then be
expressed in cells, which results in the translation
of a mutated protein; site-directed mutagenesis
can be a very valuable way to help scientists
identify critical sequences in genes that
produce proteins involved in human Diseases

A

Gene mutagenesis studies

87
Q

a technique that uses
double-stranded pieces of RNA (dsRNA) to
inhibit or silence expression of genes.

A

RNA interference

Biotechnology (2 decks)

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