Lecture 3 Reverse Transcription and complementary DNA (cDNA) Flashcards
Central Dogma
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
DNA
Genetic material, located in the nucleus
mRNA
Carries a faithful copy of a gene into the cytoplasm for protein synthesis
Proteins
Enzymatic and structural elements, make in the cytoplasm
RNA Structure
A single stranded molecule Purine Bases (double ring) Adenine and Guanine Pyrimidine Bases (single ring) Cytosine and Uracil
2 Types of RNA Bases
Purines
Pyrimidine
Purines
RNA (double ring)
Adenine and Guanine
Pyrimidines
RNA (single ring)
Cytosine and Uracil
RNA
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
Isolated RNA can be used for:
- 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
Methods to Isolate Total RNA
- Trizol Method
2. Spin column format (method we used in lab)
Trizol Method for RNA Isolation
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
Aqueous phase is:
Interphase is:
Organic phase:
Aqueous - RNA
Interphase - DNA
Organic phase - proteins, lipids
Spin column format
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
Transcription
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.
Genes in higher organisms
Are not continuous sequences: they exist on the chromosomes as seperate pieces of coding region that must be stitched together.
Exons
Coding region
The various bits of DNA that actually code for protein
Introns
The sequences that separate the exons
Transcription
During processing of mRNA, the introns are spliced out and the exons stitched together to make a single continuous coding region.
Complementary DNA
Spliced mRNA is reverse transcribed into DNA
Spicing
Removal of junk dna
RNA Polymerase
Helps to unwind
mRNA
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
% of RNA that is mRNA
1-5%
Reverse Transcription
The transcription of single stranded RNA into double stranded DNA with the help of the enzyme Reverse Transcriptase
Reverse Transcriptase
(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
Reverse Transcriptase aka
RNA directed DNA Polymerase
Reverse Transcriptase enzyme includes these two activity
DNA Polymerase
RNase H
3 Primers used for Reverse Transcription
Usually use a combo of all 3
- Sequence-specific primer
- Oligo dT primer
- Random hexamers
Sequence-specific primer
Reverse Transcription primer
Will reverse transcribe only a specific mRNA
Oligo dT primer
Reverse transcription primer
Will prime reverse transcription of any mRNA (will bind to any poly-A tail)
Random hexamers
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)
Reverse transcription applications
Understanding CA Gene expression studies Transcriptome Personal medicine How viruses can persist
Applications of RT-PCR
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
