Methods in Molecular Biology and Genetic Engineering Part 1

Question 1

utilizes laboratory-based technologies to manipulate DNA

Answer 1

genetic engineering

Question 2

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

Answer 2

Recombinant DNA

Question 3

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

Answer 3

restriction endonucleases and cloning vectors

Question 4

allow DNA to be cut into precise pieces

Answer 4

restriction endonucleases

Question 5

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

Answer 5

cloning vectors

Question 6

-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

Answer 6

nucleases

Question 7

can hydrolyze internal bonds within a polynucleotide chain

Answer 7

endonuclease

Question 8

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

Answer 8

exonuclease

Question 9

recognition site and cleavage site are the same

Answer 9

type II restriction enzymes

Question 10

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

Answer 10

Type I restriction enzymes

Question 11

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

Answer 11

Type III

Question 12

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

Answer 12

staggered cut

Question 13

cut that doesn’t leave an overhang

Answer 13

blunt double-stranded cut

Question 14

what is needed for a molecule to be cloned

Answer 14

a vector and an insert

Question 15

What should an ideal vector be able to do?

Answer 15

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

Question 16

what does a vector generally contain?

Answer 16

-selectable marker
-origin of replication
-restriction site

Question 17

Explain the cloning strategy:

Answer 17

-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

Question 18

the process by which DNA is introduced into a host cell

Answer 18

transformation

Question 19

describe the transformation process:

Answer 19

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

Question 20

cluster of identical plasmid-containing bacteria

Answer 20

colony

Question 21

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

Answer 21

TOPO cloning (Topoisomerase based cloning)

Question 22

describe the process of TOPO cloning:

Answer 22

-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
-

Question 23

describe the process of TOPO cloning:

Answer 23

-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

Question 24

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

Answer 24

blue/white screen

Question 25

describe blue/white screening for positive transformants:

Answer 25

-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

Question 26

what operon does blue/white screening utilize

Answer 26

lac

Question 27

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

Answer 27

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

Question 28

what do the blue and white colonies contain

Answer 28

-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

Question 29

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

Answer 29

expression vectors

Question 30

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

Answer 30

dual function

Question 31

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

Answer 31

Green fluorescent protein (GFP)

Question 32

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

Answer 32

fusion proteins

Question 33

gel electrophoresis

Answer 33

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

Question 34

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)

Answer 34

Agarose Gel Electrophoresis

Question 35

permits the exponential amplification of a desired sequence

Answer 35

PCR

Question 36

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

Answer 36

RT-PCR

Question 37

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

Answer 37

Real-time or quantitative PCR (qPCR)

Question 38

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

Answer 38

thermostable DNA polymerases

Question 39

What are the components of PCR?

Answer 39

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

Question 40

What are the repeated steps of PCR?

Answer 40

Denaturation, annealing, extention

Question 41

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

Answer 41

Denaturation

Question 42

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

Answer 42

Annealing

Question 43

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

Answer 43

Extension

Question 44

what does repeating the pcr step lead to

Answer 44

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

Question 45

-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

Answer 45

RT-qPCR

Question 46

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

Answer 46

Real-time PCR

Question 47

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

Answer 47

hybridization

Question 48

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

Answer 48

probe

Question 49

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

Answer 49

complementary base pairing

Question 50

-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

Answer 50

in situ hybridization (ISH)

Question 51

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

Answer 51

fluorescence in situ hybridization (FISH)

Question 52

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

Answer 52

blotting techniques

Question 53

hybridization of a probe to filter bound DNA

Answer 53

southern hybridization

Question 54

hybridization of a probe to filter bound RNA

Answer 54

Northern hybridization

Question 55

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

Answer 55

Southern Blotting

Question 56

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

Answer 56

DNA sequencing

Question 57

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

Answer 57

dideoxy sequencing or Chain termination reaction

Question 58

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)

Answer 58

Sanger Sequencing

Question 59

why is it called the chain termination reaction

Answer 59

ddNTPs terminate DNA synthesis at particular nucleotides

Question 60

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

Answer 60

ddNTPs

Question 61

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

Answer 61

ddNTPs are added in chainterm and causes extension to cease

Question 62

what is the result of chain-termination PCR

Answer 62

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

Question 63

what side of the electrode is DNA pulled to

Answer 63

positive because it is negative

Question 64

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

Answer 64

chromatogram

Question 65

what type of electrophoresis is used with Sanger sequencing

Answer 65

capillary gel

Question 66

-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

Answer 66

Chromatin Immunoprecipitation (ChIP)

Question 67

how are antibodies utilized in ChIP

Answer 67

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

Question 68

describe the process of ChIP

Answer 68

-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