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Biochemistry-Nucleic acids > Nucleic Acid Techniques > Flashcards

Nucleic Acid Techniques Flashcards

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1
Q

Nucleic Acid Techniques

A
  1. Restriction Mapping
  2. DNA Sequencing
    * Chain Terminator Procedure
    * Automated DNA Sequencing
  3. Recombinant DNA Technology
  4. Polymerase Chain Reaction (PCR)
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2
Q
  1. Restriction Mapping
A
  • Makes use of Restriction enzymes (from bacteria) to digest DNA for analysis of
    fragments generated
    -restriction maps provide framework for locating
  • base sequences
  • genes on small genomes or chromosomes
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3
Q

T or F: Bacteria have learned to “restrict” the possibility of attack from foreign DNA by means of “restriction enzymes”

A

True

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4
Q

What type of restriction enzymes cleave DNA at selected sites

A

Type II and III

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5
Q

Nucleases

A

Enzymes that hydrolyze nucleic acids

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6
Q

Endo

A

cleave at an internal location

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7
Q

Exo

A

cleave at the end

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8
Q

DNase

A

act only on DNA

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9
Q

RNase:

A

act only on RNA

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10
Q

Restriction Enzymes 3 types

A

-restriction endonucleases
-cleave dna at internal location
-Isolated chiefly from bacteria
* Type I: requires ATP, non-specific cleavage
* Type II: no ATP requirement, specific cleavage
* Type III: requires ATP, specific cleavage

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11
Q

T or F: Cleavage can leave staggered or “sticky” ends or can produce
“blunt” ends

A

true

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12
Q

The stability of the DNA double helix is due to

A
  • Hydrogen bonds – between base pairs
  • Electrostatic interactions – mutual repulsion of phosphate groups,
    which makes them most stable on the helix exterior
  • Base-pair stacking interactions
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13
Q

intercalating agents

A

Ethidium bromide
* Acridine orange
* Actinomycin D

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14
Q

Why can Intercalating Agents Distort the Double Helix

A

Because it is flexible, aromatic macrocycles – flat hydrophobic molecules
composed of fused, heterocyclic rings, can slip between the stacked pairs of bases

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15
Q

Ethidium Bromide

A

used to aid in visualization of DNA separated using
gel electrophoresis

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16
Q

Acridine Orange

A

used in staining
nucleic acids – helpful in
determining cell cycle

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17
Q

Actinomycin D

A

an antibiotic shown to have anti-cancer affects. It is shown to have the ability to inhibit transcription by binding DNA at the transcription initiation complex and preventing elongation

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18
Q

denaturation

A

When heated, DNA double helix will collapse into its two complementary
strands

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19
Q

hyperchromic effect

A

DNA denaturation results in qualitative changes in the physical properties
* Exposure of bases in denatured DNA results in an increase in absorbance

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20
Q

Tm

A

he temperature at which half of the DNA is denatured is defined as the
melting temperature

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21
Q

T OR F: DNA cannot be renatured by cooling

A

False; it can

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22
Q

reannealing

A

Renaturation requires reassociation of the DNA strands into a
double helix, a process

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23
Q

Restriction Endonucleases and Gel Analysis..

A

can be used to generate a physical map of genomes, genes, or other segments of DNA

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24
Q

Restriction maps provide a framework for locating:

A
  • base sequences
  • genes on small genomes or chromosomes.
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25
restriction–fragment length polymorphisms.
Treatment with restriction enzymes yield fragments with different lengths or what is known
26
2.DNA Sequencing
DNA Sequencing used to determine the primary structure of nucleic acids * Chain Terminator Procedure * Automated DNA Sequencing
27
* Primer extension
A template DNA base-paired with a complementary primer is copied by DNA polymerase in the presence of dATP, dCTP, dGTP, dTTP
28
Chain–terminator procedure:
1.Fragment of DNA polymerase I (Klenow fragment- polymerase activity) 2.Single Strand of DNA and a DNA primer 3.Nucleoside Triphosphate (dNTPs) – dATP, dGTP, dCTP, dTTP 4.*** small amount of 2’, 3’ dideoxynucleoside triphosphate (ddNTP)
29
ddNTP analog
Prevents addition of next nucleotide because 3’-OH needed for phosphodiester bond is replaced with a 3’-H
30
Clone:
a collection of molecules or cells, all identical to an original molecule or cell
31
T or F: Gene can be an exact or altered version of natural gene
True
32
Plasmids can be cleaved by
restriction enzymes, leaving sticky ends
33
How are artificial plasmids constructed?
by linking new DNA fragments to the sticky ends of plasmids
34
plasmid
naturally occurring, circular, extrachromosomal DNA molecules found in bacteria
35
Clone + Plasmid --> Vector
insertion of foreign DNA into artificial plasmids that serve to carry the foreign DNA insert in E.coli
36
origin of replication
a replicator
37
antibiotic resistance gene
Selectable marker
38
cloning site
site where insertion of foreign DNA will not disrupt replication or inactivate essential markers
39
molecular cloning or genetic engineering
The isolation, amplification, and modificiation of specific DNA sequences
40
Recombinant DNA
has 5 basics of cloning
41
Recombinant DNA: The 5 Basics of Cloning
1. Must be able to cut and rejoin DNA a precise locations - Must use sequence specific endonucleases called restriction endonucleases on foreign DNA and vector 2. Select a DNA molecule to serve as a carrier - this is know as the vector 3. Prepare and insert foreign DNA - Use DNA ligase to join two pieces of DNA - vector with inserted foreign DNA is known as recombinant DNA 4. Introduce vector/insert into host organism - this process is known as transformation 5. Screen for host cells replicating the hybrid DNA
42
Vector
Select a DNA molecule to serve as a carrier
43
Recombinant DNA
vector with inserted foreign DNA
44
transformation
Introduce vector/insert into host organism
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1.Plasmids
Relatively small Replicate easily Carry resistance gene(s) for antibiotics Contains multiple restriction sites
46
Biologically Functional Chimeric Plasmids : "Transformation"
the uptake and replication of exogenous DNA by a recipient cell.To facilitate transformation bacterial cells are rendered permeable to DNA by Ca+
47
An appropriate vector and clone foreign DNA
1. Plasmid 2. Bacteriophage λ 3. Bacterial artificial chromosomes (BACs)
48
2. Bacteriophage
produces large amounts of recombnant DNA in easily purified form
49
3.BAC Vectors
Bacterial artificial chromosome (BAC) vectors Allow for cloning of much larger DNA segments Replicated DNA at a level of one copy per cell
50
Recombinant DNA
Detect presence of cloned DNA 1. Antibiotic Resistance 2. Color Change 3. Radioactive Probe
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Radioactive Probe
Identification of a clone with a DNA fragment by hybridization using a radioactive probe
52
PCR( Polymerase Chain Reaction)
The small sample of DNA can serve as template for DNA polymerase, Procedure for amplifying a segment of DNA by repeated rounds of replication
53
What happens when you don’t have enough DNA for analysis?
You make more!! - PCR
54
1 Cycle has 3 temp dependent steps:
1. Denaturation- separation of DNA strands 2. Annealing- primers anneal to DNA strands 3. Extension- DNA polymerase synthesizes complementary DNA strands
55
matches the sequence of 5’-3’ template strand at the start of the DNA sequence (is complementary and therefore binds the 3’ – 5’ strand) Forward Primer: 5’- ATTAGC -3’
Forward primer
56
matches the sequence of 3’-5’ strand at the end of the DNA sequence (is complementary and therefore binds the 5’ – 3’ strand) Reverse Primer: 5’- CCCGTA -3’
Reverse primer
57
In Vitro Mutagenesis
(1) Template DNA strands are separated and amplified by PCR. (2) Following many cycles of PCR, the DNA product can be used to transform E. coli cells. (3) The plasmid DNA can be isolated and screened for the presence of the unique restriction site (by restriction endonuclease cleavage).
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page 28-31
59
Golden rice
result of an effort to develop rice varieties that produce provitamin-A (beta-carotene) as a means of alleviating vitamin A (retinol) deficiencies in the diets of poor and disadvantaged people in developing countries
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Biochemistry-Nucleic acids (2 decks)

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