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MOLBIO (LAB) > Reverse Transcription > Flashcards

Reverse Transcription Flashcards

1
Q

RNA forms a complementary strand (aka cDNA) to make a double helix structure

A

REVERSE TRANSCRIPTION

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2
Q

DNA forms mRNA to synthesize protein

A

Central dogma

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3
Q

“Dogma” means ____

A

true

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4
Q

Utilizes RNA to form complementary strand to become complementary DNA to synthesize protein

A

RNA Viruses / retrovirus

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5
Q

Examples of RNA virus/retrovirus

A

HIV
COVID-19

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6
Q

A polymer of nucleotides similar to DNA.

A

RNA

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7
Q

Sugar in RNA

A

RIbose

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8
Q

Nitrogen base in RNA

A

Uracil

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9
Q

Synthesized as a single strand

A

RNA

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10
Q

T/F: RNA becomes double stranded when there are bent

A

TRUE

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11
Q

T/F: RNA has complementary partner strands

A

FALSE; No complementary partner strands

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12
Q

○ Present in the base sequence of the RNA
○ Forms hoops or folds

A

Internal Hemologies

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13
Q

T/F: RNA can also pair with complementary single strands of DNA or another RNA to form a double helix

A

TRUE

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14
Q

Enumerate the types of RNA

A
  1. Ribosomal RNA (rRNA)
  2. Messenger RNA (mRNA)
  3. Transfer RNA (tRNA)
  4. Small Nuclear RNAs
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15
Q

rRNA is what percent of total RNA

A

80% to 90%

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16
Q

rRNA is an important structural components to _______

A

ribosomes

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17
Q

T/F: rRNA is a specific site of protein synthesis during translation

A

FALSE: Non-specific sites

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18
Q

Connection between the information stored in DNA; nucleus (transcription)

A

Messenger RNA (mRNA)

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19
Q

Messenger RNA (mRNA) is a ______

A

Translation apparatus

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20
Q

Produces the protein products responsible for the phenotype

A

Translation apparatus (mRNA)

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21
Q

Nucleic acid to protein requires reading of the mRNA

A

Transfer RNA (tRNA)

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22
Q

T/F: tRNA are relatively long, single -stranded polynucleotides

A

FALSE: relatively SHORT

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23
Q

How many baes are in tRNA?

A

73-93 bases

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24
Q

tRNA

At least ___ tRNA for each amino acid

A

one

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25
Q

Proteins are formed when:
○ _________ and _______ strand binds
○ Complement strands have amino acids and is connected by the _______

A

Complement, template

Peptide bonds

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26
Q

Copying of one strand of DNA into RNA

A

TRANSCRIPTION

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27
Q

Transcription is catalyzed by which enzyme?

A

RNA polymerase

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28
Q

Transcription occurs during which cell division phase?

A

Interphase

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29
Q

Steps in the transcription initiation:

A

(1) formation of a closed promoter complex
■ To an open promoter complex
(2) conversion of the closed promoter complex to an open promoter complex
(3) polymerizing the first few nucleotides (usually up to 10)
■ Polymerize = hybridize
(4) promoter clearance (long enough to form a stable hybrid with a template strand)

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30
Q

Directs the sequential binding of ribonucleotides

A

Elongation

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31
Q

Elongation

Directs the sequential binding of ribonucleotides happens in

A

5’ end to 3’ end

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32
Q

RNA polymerase and transcription bubble move along the template DNA

A

Elongation

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33
Q

Elongation

RNA polymerase and transcription bubble move along the template DNA

A

3’end to 5’end

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34
Q

T/F: in elongation, the melted region exposes the bases of the template DNA by pair

A

FALSE: One by one

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35
Q

________ / ___________ can signal the termination of transcription

A

Terminator / termination sites

36
Q

Specific sequences downstream of the actual gene for the RNA to be transcribed.

A

TERMINATION

37
Q

Characterized by two inverted repeats spaced

A

Intrinsic Termination

38
Q

Involves an inverted repeat (internal holologies making hairpin loop but no strings of uracil)

A

rho (ρ)—dependent termination

39
Q

internal homologies making hairpin loop but no
strings of ______

A

uracil

40
Q

Binds to the RNA during transcription and stops the RNA polymerase to stop the elongation

A

rho (ρ)

41
Q

Enzyme: reverse transcriptase (reported first by ___________)

A

Dr. Howard Temin

42
Q

Conversion of the RNA template into its complementary DNA strand (cDNA)

A

Reverse Transcription of RNA

43
Q

Essential in analysis of gene transcripts

A

Complementary DNA strand (cDNA)

44
Q

Complementary DNA strand (cDNA)

Can be ______, _______, _______ to estimate the copy of specific genes in order to characterize and validate gene expression

A

sequenced

cloned

applied

45
Q

_____________ are RNA dependent DNA polymerases

A

Reverse Trancriptases

46
Q

Bind to the RNA template and convert to complementary DNA strand with the presence of dNTP

A

Reverse Transcriptases

47
Q

What is dNTP

A

Deoxynucleotide triphosphates

48
Q

RNA must be converted to double stranded DNA first (done by the __________)

A

Reverse transcription

49
Q

T/F: Double stranded DNA is a better template for amplification than single stranded

A

TRUE

50
Q

RNA dependent DNA polymerase is derived from _____________

A

reverse transcribing viruses

51
Q

enumerate examples of reverse transcribing viruses

A

○ Mooloney Murine Leukemia Virus
○ Avian Myeblastoma Virus (AMV)

52
Q

Developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples)

A

Reverse Transcriptase PCR

53
Q

The method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR

A

Reverse Transcriptase PCR

54
Q

_________ / ________ to prime the synthesis of the initial DNA strand

A

oligodT primers

Random hexamers

55
Q

Enumerate the three kinds of primers

A

a. Oligo dTs
b. Gene specific primers
c. Random hexamers

56
Q

Highly specific for the target of interest

A

Oligo dTs

57
Q

Poly “T” sequences: ______ bases long that will prime complementary DNA synthesis only from RNA with _____________

A

18

poly A tails

58
Q

End part of RNa with multiple adenine bases

A

poly A tails

59
Q

End part of RNA with multiple thymine bases

A

Poly T tails

60
Q

Synthetic DNA strands which can be represented as a sequence of ‘T’ nucleotides

A

Oligo dTs

61
Q

Can be designed to anneal to a specific gene

A

Gene specific primers

62
Q

T/F: Gene specific primers has a specific base pair

A

TRUE

63
Q

Random hexamers aka ______

A

Decamers

64
Q

6-10 long base with random bases

A

Random hexamers/decamers

65
Q

Match and hybridize the random site of target RNA to prime DNA synthesis

A

Random hexamers/decamers

66
Q

Random hexamers consist of how many bases which anneal ______ to RNA molecules?

A

10 bases

randomly

67
Q

Enumerate enzymes employed for reverse transcriptase

A

Reverse Transcriptase
Rnase H
DNA Polymerase I (Klenow Fragment)

68
Q

ENZYME

○ cDNA strand
○ Still has errors

A

Reverse Transcriptase

69
Q

An enzyme that hydrolyzes RNA from a complementary DNA strand

A

Rnase H

70
Q

polymerase activity; If without the small fragment: klenow fragment used in laboratories

A

Long fragment

71
Q

Exonuclease activity

A

Small fragment

72
Q

Enumerate the steps/process of RT-PCR

A

STEP 1: ANNEALING OF THE PRIMER
STEP 2: REVERSE TRANSCRIPTION
STEP 3: REMOVAL OF MISMATCHES BY RNASE H
STEP 4: REPAIR OF STRAND BY DNA POLYMERASE I KF

73
Q

STEP 1: ANNEALING OF THE PRIMER

RNA tends to form secondary structures via ____________________________ (AT/U & CC)

A

Watson-Crick base pairing

74
Q

STEP 1: ANNEALING OF THE PRIMER

In order to remove the secondary structure

A

Denaturation

75
Q

Samples are heated with the gene specific primer
Then, cooling at room temperature

A

STEP 1: ANNEALING OF THE PRIMER

76
Q

STEP 1: ANNEALING OF THE PRIMER

Cooling at room temperature facilitates __________

A

annealing

77
Q

Reverse transcriptase commences transcription at the primer binding site

A

STEP 2: REVERSE TRANSCRIPTION

78
Q

Usual errors are the uracils (it should be thymine as it is a cDNA)

A

STEP 3: REMOVAL OF MISMATCHES BY RNASE H

79
Q

STEP 3: REMOVAL OF MISMATCHES BY RNASE H

Usual errors are the _________

A

Uracils

80
Q

Errors are filled by the DNA polymerase or the Klenow Fragment

A

STEP 4: REPAIR OF STRAND BY DNA POLYMERASE I KF

81
Q

Errors are filled by the DNA polymerase or the Klenow Fragment

A

STEP 4: REPAIR OF STRAND BY DNA POLYMERASE I KF

82
Q

The resulting DNA strand can be subjected to DNA sequencing in order to determine the identity of the transcript

A

DOWNSTREAMING PROCESS

83
Q

Can also be amplifying: multiplication of the strands

A

DOWNSTREAMING PROCESS

84
Q

Simplicity and convenience and can minimize chance of contamination

A

One step RT PCR:

85
Q

Cannot be used for detecting multiple products

A

One step RT PCR

86
Q

Two step RT PCR steps:

A

○ 1st step: RT reaction (product: cDNA)
○ 2nd step: PCR amplification

MOLBIO (LAB) (3 decks)

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