Reverse Transcription Flashcards
1
Q
RNA forms a complementary strand (aka cDNA) to make a double helix structure
A
REVERSE TRANSCRIPTION
2
Q
DNA forms mRNA to synthesize protein
A
Central dogma
3
Q
“Dogma” means ____
A
true
4
Q
Utilizes RNA to form complementary strand to become complementary DNA to synthesize protein
A
RNA Viruses / retrovirus
5
Q
Examples of RNA virus/retrovirus
A
HIV
COVID-19
6
Q
A polymer of nucleotides similar to DNA.
A
RNA
7
Q
Sugar in RNA
A
RIbose
8
Q
Nitrogen base in RNA
A
Uracil
9
Q
Synthesized as a single strand
A
RNA
10
Q
T/F: RNA becomes double stranded when there are bent
A
TRUE
11
Q
T/F: RNA has complementary partner strands
A
FALSE; No complementary partner strands
12
Q
○ Present in the base sequence of the RNA
○ Forms hoops or folds
A
Internal Hemologies
13
Q
T/F: RNA can also pair with complementary single strands of DNA or another RNA to form a double helix
A
TRUE
14
Q
Enumerate the types of RNA
A
- Ribosomal RNA (rRNA)
- Messenger RNA (mRNA)
- Transfer RNA (tRNA)
- Small Nuclear RNAs
15
Q
rRNA is what percent of total RNA
A
80% to 90%
16
Q
rRNA is an important structural components to _______
A
ribosomes
17
Q
T/F: rRNA is a specific site of protein synthesis during translation
A
FALSE: Non-specific sites
18
Q
Connection between the information stored in DNA; nucleus (transcription)
A
Messenger RNA (mRNA)
19
Q
Messenger RNA (mRNA) is a ______
A
Translation apparatus
20
Q
Produces the protein products responsible for the phenotype
A
Translation apparatus (mRNA)
21
Q
Nucleic acid to protein requires reading of the mRNA
A
Transfer RNA (tRNA)
22
Q
T/F: tRNA are relatively long, single -stranded polynucleotides
A
FALSE: relatively SHORT
23
Q
How many baes are in tRNA?
A
73-93 bases
24
Q
tRNA
At least ___ tRNA for each amino acid
A
one
25
Proteins are formed when:
○ _________ and _______ strand binds
○ Complement strands have amino acids and is connected by the _______
Complement, template
Peptide bonds
26
Copying of one strand of DNA into RNA
TRANSCRIPTION
27
Transcription is catalyzed by which enzyme?
RNA polymerase
28
Transcription occurs during which cell division phase?
Interphase
29
Steps in the transcription initiation:
(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)
30
Directs the sequential binding of ribonucleotides
Elongation
31
Elongation
Directs the sequential binding of ribonucleotides happens in
5' end to 3' end
32
RNA polymerase and transcription bubble move along the template DNA
Elongation
33
Elongation
RNA polymerase and transcription bubble move along the template DNA
3'end to 5'end
34
T/F: in elongation, the melted region exposes the bases of the template DNA by pair
FALSE: One by one
35
________ / ___________ can signal the termination of transcription
Terminator / termination sites
36
Specific sequences downstream of the actual gene for the RNA to be transcribed.
TERMINATION
37
Characterized by two inverted repeats spaced
Intrinsic Termination
38
Involves an inverted repeat (internal holologies making hairpin loop but no strings of uracil)
rho (ρ)—dependent termination
39
internal homologies making hairpin loop but no
strings of ______
uracil
40
Binds to the RNA during transcription and stops the RNA polymerase to stop the elongation
rho (ρ)
41
Enzyme: reverse transcriptase (reported first by ___________)
Dr. Howard Temin
42
Conversion of the RNA template into its complementary DNA strand (cDNA)
Reverse Transcription of RNA
43
Essential in analysis of gene transcripts
Complementary DNA strand (cDNA)
44
Complementary DNA strand (cDNA)
Can be ______, _______, _______ to estimate the copy of specific genes in order to characterize and validate gene expression
sequenced
cloned
applied
45
_____________ are RNA dependent DNA polymerases
Reverse Trancriptases
46
Bind to the RNA template and convert to complementary DNA strand with the presence of dNTP
Reverse Transcriptases
47
What is dNTP
Deoxynucleotide triphosphates
48
RNA must be converted to double stranded DNA first (done by the __________)
Reverse transcription
49
T/F: Double stranded DNA is a better template for amplification than single stranded
TRUE
50
RNA dependent DNA polymerase is derived from _____________
reverse transcribing viruses
51
enumerate examples of reverse transcribing viruses
○ Mooloney Murine Leukemia Virus
○ Avian Myeblastoma Virus (AMV)
52
Developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples)
Reverse Transcriptase PCR
53
The method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR
Reverse Transcriptase PCR
54
_________ / ________ to prime the synthesis of the initial DNA strand
oligodT primers
Random hexamers
55
Enumerate the three kinds of primers
a. Oligo dTs
b. Gene specific primers
c. Random hexamers
56
Highly specific for the target of interest
Oligo dTs
57
Poly “T” sequences: ______ bases long that will prime complementary DNA synthesis only from RNA with _____________
18
poly A tails
58
End part of RNa with multiple adenine bases
poly A tails
59
End part of RNA with multiple thymine bases
Poly T tails
60
Synthetic DNA strands which can be represented as a sequence of ‘T’ nucleotides
Oligo dTs
61
Can be designed to anneal to a specific gene
Gene specific primers
62
T/F: Gene specific primers has a specific base pair
TRUE
63
Random hexamers aka ______
Decamers
64
6-10 long base with random bases
Random hexamers/decamers
65
Match and hybridize the random site of target RNA to prime DNA synthesis
Random hexamers/decamers
66
Random hexamers consist of how many bases which anneal ______ to RNA molecules?
10 bases
randomly
67
Enumerate enzymes employed for reverse transcriptase
Reverse Transcriptase
Rnase H
DNA Polymerase I (Klenow Fragment)
68
ENZYME
○ cDNA strand
○ Still has errors
Reverse Transcriptase
69
An enzyme that hydrolyzes RNA from a complementary DNA strand
Rnase H
70
polymerase activity; If without the small fragment: klenow fragment used in laboratories
Long fragment
71
Exonuclease activity
Small fragment
72
Enumerate the steps/process of RT-PCR
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
STEP 1: ANNEALING OF THE PRIMER
RNA tends to form secondary structures via ____________________________ (AT/U & CC)
Watson-Crick base pairing
74
STEP 1: ANNEALING OF THE PRIMER
In order to remove the secondary structure
Denaturation
75
Samples are heated with the gene specific primer
Then, cooling at room temperature
STEP 1: ANNEALING OF THE PRIMER
76
STEP 1: ANNEALING OF THE PRIMER
Cooling at room temperature facilitates __________
annealing
77
Reverse transcriptase commences transcription at the primer binding site
STEP 2: REVERSE TRANSCRIPTION
78
Usual errors are the uracils (it should be thymine as it is a cDNA)
STEP 3: REMOVAL OF MISMATCHES BY RNASE H
79
STEP 3: REMOVAL OF MISMATCHES BY RNASE H
Usual errors are the _________
Uracils
80
Errors are filled by the DNA polymerase or the Klenow Fragment
STEP 4: REPAIR OF STRAND BY DNA POLYMERASE I KF
81
Errors are filled by the DNA polymerase or the Klenow Fragment
STEP 4: REPAIR OF STRAND BY DNA POLYMERASE I KF
82
The resulting DNA strand can be subjected to DNA sequencing in order to determine the identity of the transcript
DOWNSTREAMING PROCESS
83
Can also be amplifying: multiplication of the strands
DOWNSTREAMING PROCESS
84
Simplicity and convenience and can minimize chance of contamination
One step RT PCR:
85
Cannot be used for detecting multiple products
One step RT PCR
86
Two step RT PCR steps:
○ 1st step: RT reaction (product: cDNA)
○ 2nd step: PCR amplification
