Molecular Biology: mRNA to DNA

Reverse Transcription and cDNA

• mRNA is produced when genes are transcribed, but it does not contain introns
• To express eukaryotic proteins or identify genes expressed in a particular cell type, we need to make DNA copies of the transcribed mRNA
• Complementary DNA (cDNA) is a DNA copy of a mRNA, produced using an enzyme called reverse transcriptase
• cDNA libraries contain complementary DNA copies of the mRNAs present in a cell population and represent the genes being expressed in the population from where the mRNA is harvested
• cDNAs are used as the introns are removed and the clone size is smaller

DNA Sequencing

• Full characterisation of genes within the genome can be obtained by sequencing our DNA
• This provides the sequence of bases which can be used to predict the sequence of amino acids contained in the protein that the gene encodes
• DNA sequence from different individuals can also allow us to discover genetic differences, the first step to personalized medicine
• DNA sequencing method devised by Sanger is the most common method (Nobel Prize 1980)
• Uses DNA polymerase to copy single-stranded DNA
• Makes use of ‘dideoxy’ nucleotides which interrupt the ability of DNA polymerase to copy DNA

Dideoxynucleotides

• DNA sequencing uses dideoxynucleotides (ddNTPs) which do not have a –OH on the 3’ carbon
• DNA polymerase cannot incorporate any further nucleotides after incorporation of a ddNTP as no 3’ OH to form phosphodiester bond
• Dideoxynucleotide sequencing terminates replication
• Automated DNA sequencing uses a single reaction in which all four ddNTPs are included together with dNTPs
• Each ddNTP is labeled with a unique fluorescent marker

PCR (Polymerase Chain Reaction)

• Allows amplification of specific DNA sequences
• PCR reactions performed in Thermocycler
• Set machine to cycle through the PCR steps: denaturation, anneal, and extension
• PCR products: amplified DNA fragments are separated by gel electrophoresis
• PCR product size equal to the amount of DNA between the primers
• Uses of PCR:
  • Amplify whole genes or parts of genes from genomic DNA or cDNA for use in cloning and molecular analysis
  • Amplify specific regions of DNA from variety of samples, e.g. specific gene in different individuals to perform genetic diagnosis
  • Amplify DNA from sources where DNA is limited, e.g. blood spots, fossils
  • Identify victims in natural disasters, major accidents, e.g. World Trade Center

Applications of PCR

• PCR is routinely used in screening of mutations involved in genetic disorders
• PCR is a key diagnostic methodology for the detection and identification of bacteria and viruses in humans
• Identify pathogenic bacteria in contaminated food
• PCR techniques are used to analyze samples from single cells
• PCR used in forensics to identify individuals from body fluids left at a crime scene or in paternity testing
• Used to check and confirm DNA constructs or transgenic animals

DNA Microarrays

• Modern devices that use nucleic acid hybridization to rapidly measure which genes are expressed in a tissue sample
• Nucleic acid hybridization occurs when complementary strands of DNA anneal to one another
• Enables the ability to simultaneously identify all the genes expressed in a particular sample
• Uses an approach whereby all the mRNA from a sample is converted to cDNA, fluorescently labeled, denatured into single strands, and used as probe
• This probe is applied to the array which contains spots where single-stranded DNA from each gene in the genome is laid out in an array

Learn about the process of converting mRNA back to DNA through reverse transcription and its importance in understanding gene expression in different cells and diseases.