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Molecular Biology > Methods in Molecular Biology and Genetic Engineering Part 1 > Flashcards

Methods in Molecular Biology and Genetic Engineering Part 1 Flashcards

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1
Q

utilizes laboratory-based technologies to manipulate DNA

A

genetic engineering

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2
Q

used as a tool to analyze gene structure and expression, changes in the DNA content of bacteria and eukaryotic cells by directly introducing cloned DNA to be apart of the genome

A

Recombinant DNA

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3
Q

what are the 2 essentials of a molecular biologist’s toolkit?

A

restriction endonucleases and cloning vectors

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4
Q

allow DNA to be cut into precise pieces

A

restriction endonucleases

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5
Q

such as plasmids or phages used to carry inserted foreign DNA fragments

A

cloning vectors

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6
Q

-enzymes that degrade nucleic acids, opposite of polymerase
-hydrolyze or break an ester bond in a phosphodiester linkage between adjacent nucleotides in a polynucleotide chain

A

nucleases

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7
Q

can hydrolyze internal bonds within a polynucleotide chain

A

endonuclease

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8
Q

acts at the end of a chain and hydrolyze from that end position

A

exonuclease

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9
Q

recognition site and cleavage site are the same

A

type II restriction enzymes

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10
Q

cleavage site can be up to 1000 bp away from the recognition site

A

Type I restriction enzymes

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11
Q

closer cleavage sites, usually up to 20 to 30 bp away from the recognition site

A

Type III

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12
Q

cut which leaves single-stranded 3’ or a 5’ overhang, that easily re-attach to other ends (sticky ends)

A

staggered cut

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13
Q

cut that doesn’t leave an overhang

A

blunt double-stranded cut

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14
Q

what is needed for a molecule to be cloned

A

a vector and an insert

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15
Q

What should an ideal vector be able to do?

A

replicate autonomously and be able to amplify insert DNA up to 10 kb

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16
Q

what does a vector generally contain?

A

-selectable marker
-origin of replication
-restriction site

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17
Q

Explain the cloning strategy:

A

-restriction enzyme cuts vector and insert
-vector and insert are combined
-complementary overhangs are fused together with T4 DNA ligase
-ligated molecules are used to transform E.coli

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18
Q

the process by which DNA is introduced into a host cell

A

transformation

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19
Q

describe the transformation process:

A

-competent E.coli take up outside DNA
-bacteria placed on antibiotic plates to see which ones took up a plasmid
-colonies form

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20
Q

cluster of identical plasmid-containing bacteria

A

colony

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21
Q

cloning that relies on the hybridization of the complementary base pairs Adenine and Thymine

A

TOPO cloning (Topoisomerase based cloning)

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22
Q

describe the process of TOPO cloning:

A

-Taq polymerase leaves a single A overhang on the 3’ end of PCR products or insert
-Thymine comes from a pre-cut , linear cloning ready TOPO vector that has a DNA topoisomerase I fused to the 3’ end
-topoisomerase acts as a ligase & joins A&T together
-

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23
Q

describe the process of TOPO cloning:

A

-Taq polymerase leaves a single A overhang on the 3’ end of PCR products or insert
-Thymine comes from a pre-cut , linear cloning ready TOPO vector that has a DNA topoisomerase I fused to the 3’ end
-topoisomerase acts as a ligase & joins A&T together

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24
Q

screening technique that allows for quick and easy detection of successful ligations in vector-based gene cloning

A

blue/white screen

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25
Q

describe blue/white screening for positive transformants:

A

-DNA is ligated into a vector
-vector is transformed into competent cells that are grow in the presence of X-gal
-success=white colony; unsuccessful=blue

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26
Q

what operon does blue/white screening utilize

A

lac

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27
Q

How is the presence of an active B-galactosidase detected by X-gal within the agar plate?

A

X-gal is cleaved by B-galactosidase to form a bright blue insoluble pigment

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28
Q

what do the blue and white colonies contain

A

-blue contain the vector without an insert because the B-gal cleaved Xgal into a blue compund
-white remains colorless bc the inactivated B-gal did not cleave X-gal

29
Q

what vectors are used to clone larger inserts, express cloned genes in cells, investigate properties of a promoter or create various fusion proteins?

A

expression vectors

30
Q

introduction of gene of interest into the host cell and aids in the analysis of the foreign gene via relevant protein product expression

A

dual function

31
Q

-a naturally fluorescent protein that, when excited with one wavelength of light, emits fluorescence in another wavelength
-used to visualize patterns of gene expression

A

Green fluorescent protein (GFP)

32
Q

a protein of interest is fused to GFP and can thus be visualized in living tissues

A

fusion proteins

33
Q

gel electrophoresis

A

separates DNA fragments by size, using an electric current to cause DNA to migrate toward a positive charge; smaller fragments migrate faster

34
Q

electrophoresis is done by preparing a small slab of gel in an electrically conductive mildly basic buffer; negatively charged DNA moves by electric current (smaller moves faster)

A

Agarose Gel Electrophoresis

35
Q

permits the exponential amplification of a desired sequence

A

PCR

36
Q

uses reverse transcriptase to convert RNA to DNA for use in a PCR

A

RT-PCR

37
Q

detects the products of PCR amplification during their synthesis and is more sensitive and quantitative than conventional PCR

A

Real-time or quantitative PCR (qPCR)

38
Q

PCR depends on the use of _______________________ that can withstand multiple cycles of template denaturation

A

thermostable DNA polymerases

39
Q

What are the components of PCR?

A

-DNA template
-DNA polymerase
-Primers
-Nucleotides (dNTPs)

40
Q

What are the repeated steps of PCR?

A

Denaturation, annealing, extention

41
Q

the two strands of DNA are separated; normally done at 94 C

A

Denaturation

42
Q

the primers bind to the DNA; normally done at 55 C

A

Annealing

43
Q

using the DNA template, the polymerase adds the dNTPs to the primer and causes the extension of the prime; normally done at 72 C

A

Extension

44
Q

what does repeating the pcr step lead to

A

N(thermal cycles) exponential increase (2N) allowing for phenomenal levels of amplification

45
Q

-combines the effects of reverse transcription and quantitative PCR to amplify and detect specific targets
-variety of applications including quantifying gene expression levels, validating RNAi, and detecting pathogen

A

RT-qPCR

46
Q

allows collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step
-detection based on fluorescence technology

A

Real-time PCR

47
Q

-detection of nucleic acid to complementary sequences
-involves the bonding of a probe to a target seqence

A

hybridization

48
Q

short, complementary nucleic acid strand
-generally labeled with a radioisotope or fluorescent molecule

A

probe

49
Q

a positive hybridization signal is obtained when _____________ occurs between the probe and the target sequence

A

complementary base pairing

50
Q

-allows the detection and localization of viral nucleic acid in tissue sections or cytological specimens using labelled nucleic acid probes with complementary sequences to the target viral nucleic acid

A

in situ hybridization (ISH)

51
Q

uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope

A

fluorescence in situ hybridization (FISH)

52
Q

commonly used for nucleic acid detection following the hybridization of a radioactive probe to filter bound DNA or RNA

A

blotting techniques

53
Q

hybridization of a probe to filter bound DNA

A

southern hybridization

54
Q

hybridization of a probe to filter bound RNA

A

Northern hybridization

55
Q

involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridization with a labeled probe

A

Southern Blotting

56
Q

involves determination of nucleotide bases A,T,G and C in the DNA

A

DNA sequencing

57
Q

The classic method of DNA sequencing called _____________________________________ has been significantly used since its discovery by Frederick Sanger and colleagues in 1977.

A

dideoxy sequencing or Chain termination reaction

58
Q

method that requires many identical copies of DNA, an oligonucleotide primer that is complementary to a short stretch of the DNA, DNA polymerase, deoxynucleotides, and dideoxynucleotides (ddNTPS)

A

Sanger Sequencing

59
Q

why is it called the chain termination reaction

A

ddNTPs terminate DNA synthesis at particular nucleotides

60
Q

modified nucleotides that can be incorporated into the growing DNA strand but lack the 3’ hydroxyl group needed to attach the next nucleotide

A

ddNTPs

61
Q

what is the difference between chain-termination PCR and standard PCR

A

ddNTPs are added in chainterm and causes extension to cease

62
Q

what is the result of chain-termination PCR

A

millions to billions of oligonucleotide copies of the DNA sequence of interest, terminated at random lengths (n) by 5’-ddNTPS

63
Q

what side of the electrode is DNA pulled to

A

positive because it is negative

64
Q

shows the fluorescent peak of each nucleotide along the length of the template

A

chromatogram

65
Q

what type of electrophoresis is used with Sanger sequencing

A

capillary gel

66
Q

-allows detection of specific protein-DNA interactions in vivo and mapping of all the protein-binding sites for a given protein across the entire genome
-used to identify the relative abundance of a specific protein or specific protein modification at a certain region in the genome

A

Chromatin Immunoprecipitation (ChIP)

67
Q

how are antibodies utilized in ChIP

A

antibodies selectively recognize and bind proteins, including histones, histone modifications, transcription factors, and cofactors to provide information about chromatin states and gene transcription

68
Q

describe the process of ChIP

A

-protein and DNA are crosslinked
-chromatin broken into small fragments
-antibody used to immunoprecipitate protein of interest
-DNA then purified and analyzed by PCR, sequencing, or by labeling the DNA and applying to a tiling array to detect genome-wide interactions

Molecular Biology (8 decks)

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