Lecture 3 Reverse Transcription and complementary DNA (cDNA)

Question 1

Central Dogma

Answer 1

Information in nucleic acid can be perpetuated or transferred, but the transfer of information form into protein is irreversible

DNA is transcribed to RNA is translated to Proteins

Question 2

DNA

Answer 2

Genetic material, located in the nucleus

Question 3

mRNA

Answer 3

Carries a faithful copy of a gene into the cytoplasm for protein synthesis

Question 4

Proteins

Answer 4

Enzymatic and structural elements, make in the cytoplasm

Question 5

RNA Structure

Answer 5

A single stranded molecule
Purine Bases (double ring) Adenine and Guanine
Pyrimidine Bases (single ring) Cytosine and Uracil

Question 6

2 Types of RNA Bases

Answer 6

Purines

Pyrimidine

Question 7

Purines

Answer 7

RNA (double ring)

Adenine and Guanine

Question 8

Pyrimidines

Answer 8

RNA (single ring)

Cytosine and Uracil

Question 9

RNA

Answer 9

80-85% of total RNA is ribosomal RNA

Remaining 15-29% consists of low-molecular weight species, such as transfer RNA and small nuclear RNA

Question 10

Isolated RNA can be used for:

Answer 10

• poly(A+)(mRNA) selection
• Northern hybridization
• In vitro translation (IVT) - in vitro protein synthesis
• Expression analysis - RNase Protection Assay
• Reverse Transcription (RT)
• Transcriptome/Expression analysis

Question 11

Methods to Isolate Total RNA

Answer 11

1. Trizol Method

2. Spin column format (method we used in lab)

Question 12

Trizol Method for RNA Isolation

Answer 12

Lysis of cells with Trizol (monophasic solution of guanidine isothiocyanate and phenol)
Addition of chloroform generates second (organic) phase into which DNA and proteins are extracted, leaving RNA in the aqueous supernatant

Question 13

Aqueous phase is:
Interphase is:
Organic phase:

Answer 13

Aqueous - RNA
Interphase - DNA
Organic phase - proteins, lipids

Question 14

Spin column format

Answer 14

Based on the fact of RNA selective binding properties of a silica gel based membrane under high salt buffer:
1. Lyse and homogenize cells in presence of gaunidine isothiocyanate-containing buffer
2. Add homogenate to column with a silica-based membrane in the presence of thanol
3. Wash column
4. Elute RNA
Advantage - there is nothing toxic about this protocol

Question 15

Transcription

Answer 15

Process by which a DNA sequence is copied to produce a complementary RNA
The transfer of genetic info from DNA into RNA
Beginning of the process that ultimately leads to the translation of the genetic code (via mRNA) into a protein.

Question 16

Genes in higher organisms

Answer 16

Are not continuous sequences: they exist on the chromosomes as seperate pieces of coding region that must be stitched together.

Question 17

Exons

Answer 17

Coding region

The various bits of DNA that actually code for protein

Question 18

Introns

Answer 18

The sequences that separate the exons

Question 19

Transcription

Answer 19

During processing of mRNA, the introns are spliced out and the exons stitched together to make a single continuous coding region.

Question 20

Complementary DNA

Answer 20

Spliced mRNA is reverse transcribed into DNA

Question 21

Spicing

Answer 21

Removal of junk dna

Question 22

RNA Polymerase

Answer 22

Helps to unwind

Question 23

mRNA

Answer 23

Messenger RNA
a large family of RNA molecules that convey genetic info from DNA to the ribosome, where they specify the amino acid sequence of the nascent proteins
1-5% of the total cellular RNA

Question 24

% of RNA that is mRNA

Answer 24

1-5%

Question 25

Reverse Transcription

Answer 25

The transcription of single stranded RNA into double stranded DNA with the help of the enzyme Reverse Transcriptase

Question 26

Reverse Transcriptase

Answer 26

(RT)
Transcribes single stranded mRNA into double stranded DNA
Complex molecular machines with moving parts and multiple activities
Common in retrovirus -HIV -M-MLV (Moloney Murine Leukemia Virus) -AMV (Avian Myeloblastosis Virus)
Includes two activities: DNA polymerase and RNase H

Question 27

Reverse Transcriptase aka

Answer 27

RNA directed DNA Polymerase

Question 28

Reverse Transcriptase enzyme includes these two activity

Answer 28

DNA Polymerase

RNase H

Question 29

3 Primers used for Reverse Transcription

Answer 29

Usually use a combo of all 3

1. Sequence-specific primer
2. Oligo dT primer
3. Random hexamers

Question 30

Sequence-specific primer

Answer 30

Reverse Transcription primer

Will reverse transcribe only a specific mRNA

Question 31

Oligo dT primer

Answer 31

Reverse transcription primer

Will prime reverse transcription of any mRNA (will bind to any poly-A tail)

Question 32

Random hexamers

Answer 32

Reverse transcription primer
A mixture of 6nt long primers containing all possible sequences of 6nt; these will hybridize all over the place and prime reverse transcription of any RNA (in various sizes)

Question 33

Reverse transcription applications

Answer 33

Understanding CA
Gene expression studies
Transcriptome
Personal medicine
How viruses can persist

Question 34

Applications of RT-PCR

Answer 34

1st and most imp personalize medicine Ex. Breast CA
Cloning genes’ expressed forms (not genomic version)
Monitor a gene’s expression level in any tissue
Sophisticated RT-PCR: The real time PCR
Sequencing a whole mRNA profile
Microarray analysis (DNA chip)
Diagnose and easily differentiate between different cancer types
Early detection of hidden illnesses