Molecular Biology: mRNA to DNA

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Questions and Answers

What is the primary advantage of using cDNA instead of genomic DNA for cloning?

It increases the complexity of the DNA sequence

It produces a smaller clone size (correct)

It allows for the expression of non-coding regions

It eliminates the need for reverse transcription

What is the main purpose of creating a cDNA library?

To represent the genes being expressed in a cell population (correct)

To study the regulation of gene expression

To analyze the structure of genomic DNA

To identify genes that are not expressed in a particular cell type

What is the primary function of reverse transcriptase in cDNA synthesis?

To amplify DNA sequences

To translate mRNA into protein

To remove introns from genomic DNA

To convert mRNA into DNA (correct)

Why is it necessary to harvest mRNA from different cells?

To determine which genes are expressed by which cells and how this changes in disease Signup and view all the answers

What is the benefit of using DNA sequencing to characterize genes?

It provides the sequence of bases which can be used to predict the sequence of amino acids Signup and view all the answers

What is the primary application of DNA cloning in biotechnology?

To produce recombinant proteins for therapeutic use Signup and view all the answers

What is the primary purpose of using ddNTPs in automated DNA sequencing?

To label each fragment with a unique fluorescent marker Signup and view all the answers

How do automated DNA sequencers distinguish between different ddNTPs?

By their different fluorescent colors Signup and view all the answers

What is the primary advantage of Next Generation Sequencing (NGS) over previous sequencing technologies?

Ability to sequence thousands to millions of DNA fragments per day Signup and view all the answers

What is the estimated number of protein-coding genes in the human genome?

30,000 Signup and view all the answers

What is the primary purpose of separating DNA fragments on a capillary gel?

To separate the fragments based on their size Signup and view all the answers

What is the average read length of fluorescent capillary dideoxy sequencing?

800-1000bp Signup and view all the answers

What is the primary difference between the original radioactive dideoxy sequencing and fluorescent capillary dideoxy sequencing?

The method of detection Signup and view all the answers

What is the advantage of Next Generation Sequencing (NGS) over fluorescent capillary dideoxy sequencing?

Faster sequencing times Signup and view all the answers

What is the primary purpose of DNA sequencing?

To identify genetic differences between individuals Signup and view all the answers

What is the role of dideoxynucleotides in DNA sequencing?

To terminate the replication of DNA at specific points Signup and view all the answers

What is the difference between deoxynucleotides and dideoxynucleotides?

Deoxynucleotides have a 3' OH group, while dideoxynucleotides do not Signup and view all the answers

What is the result of incorporating a dideoxynucleotide into a DNA strand?

The DNA strand is terminated at a specific point Signup and view all the answers

What is the purpose of using dideoxynucleotides in the Sanger sequencing method?

To terminate the replication of DNA at specific points Signup and view all the answers

What is the result of using a mixture of dCTP and ddCTP in DNA sequencing?

The DNA strand is terminated at random points Signup and view all the answers

Why is the Sanger sequencing method also known as the dideoxynucleotide chain-termination sequencing method?

Because it uses dideoxynucleotides to terminate the replication of DNA Signup and view all the answers

What is the advantage of the Sanger sequencing method?

It can be automated and allows rapid sequencing of large amounts of DNA Signup and view all the answers

What is the primary purpose of reducing the reaction temperature to 45-68°C during the PCR process?

To allow primers to hybridize to their complementary sequences in the target DNA Signup and view all the answers

What is the temperature range for primer extension during the PCR process?

72°C Signup and view all the answers

What is the purpose of repeating the PCR cycle 30 times?

To increase the yield of amplified DNA fragments Signup and view all the answers

What is the primary application of PCR in genetic diagnosis?

To amplify specific regions of DNA from variety of samples Signup and view all the answers

What is the purpose of using PCR in forensic analysis?

To identify individuals from body fluids left at a crime scene Signup and view all the answers

What is the primary function of DNA microarrays?

To rapidly measure which genes are expressed in a tissue sample Signup and view all the answers

What is the temperature used for denaturation during the PCR process?

96°C Signup and view all the answers

What is the purpose of gel electrophoresis in PCR?

To separate amplified DNA fragments by size Signup and view all the answers

What is the primary function of nucleic acid hybridisation in the context of DNA microarray?

To simultaneously identify all the genes expressed in a particular sample Signup and view all the answers

What is the purpose of converting mRNA into cDNA in the DNA microarray method?

To create a single-stranded DNA probe Signup and view all the answers

What is the role of the chip reader in the DNA microarray method?

To detect the fluorescent spots on the microarray Signup and view all the answers

What is the outcome of the hybridization process in the DNA microarray method?

The labelled cDNA will bind to the spots on the microarray that contain complementary sequences Signup and view all the answers

What is the purpose of creating an array of single-stranded DNA on the microarray slide?

To provide a platform for the hybridization of the labelled cDNA probe Signup and view all the answers

What is the relationship between the brightness of the fluorescent spots on the microarray and the level of gene expression?

The brightness of the fluorescent spots is directly proportional to the level of gene expression Signup and view all the answers

Study Notes

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

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Description

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.