Molecular Biology: Gene Expression and cDNA

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Questions and Answers

What is the primary reason for creating cDNA from mRNA?

To express prokaryotic proteins

To clone RNA molecules

To study the structure of introns

To identify genes expressed in a particular cell type (correct)

What is the main advantage of using cDNA libraries over genomic DNA?

cDNA libraries contain more introns

cDNA libraries are more difficult to sequence

cDNA libraries are larger in size

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

What is the enzyme used to produce cDNA from mRNA?

DNA polymerase

Reverse transcriptase (correct)

Protease

RNA polymerase

What is the primary application of DNA sequencing?

Full characterization of genes within the genome Signup and view all the answers

Why is it necessary to remove introns from mRNA?

To make DNA copies of the mRNA Signup and view all the answers

What is the primary difference between genomic DNA and cDNA?

cDNA is a DNA copy of a mRNA Signup and view all the answers

What is the primary purpose of DNA sequencing in the context of personalized medicine?

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

What is the role of DNA polymerase in the Sanger sequencing method?

To copy single-stranded DNA Signup and view all the answers

What is the characteristic feature of dideoxynucleotides that allows them to terminate DNA replication?

Absence of a 3' OH group Signup and view all the answers

What determines the length of the fragments in dideoxynucleotide sequencing?

The time of ddCTP incorporation Signup and view all the answers

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

The DNA polymerase stops extending the chain Signup and view all the answers

What is the purpose of using dideoxynucleotides in DNA sequencing?

To terminate DNA replication Signup and view all the answers

What is the name of the sequencing method devised by Sanger?

Dideoxynucleotide chain-termination sequencing 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 primary purpose of using fluorescent markers in automated DNA sequencing?

To identify which ddNTP was added at the end of each fragment 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 Next Generation Sequencing (NGS) and traditional dideoxy sequencing?

The sequential addition of nucleotides on microchips Signup and view all the answers

What was the original read length of radioactive dideoxy sequencing on gels?

200-400bp 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 purpose of the laser light in automated DNA sequencing?

To excite the fluorescent tag on each fragment Signup and view all the answers

What is the primary advantage of Next Generation Sequencing (NGS) over traditional dideoxy sequencing?

Rapid sequencing of thousands to millions of DNA fragments Signup and view all the answers

What is the average amount of DNA that can be sequenced per hour using fluorescent capillary dideoxy sequencing?

10-20,000bp Signup and view all the answers

What is the temperature range for primer annealing in PCR?

58-68°C Signup and view all the answers

What is the purpose of the 96°C step in PCR?

Denaturation Signup and view all the answers

What is the maximum size of DNA fragments that can be amplified using PCR?

5kb Signup and view all the answers

What is the primary application of PCR in genetic diagnosis?

Amplifying specific regions of DNA Signup and view all the answers

What is the purpose of PCR in forensic analysis?

Identifying individuals from body fluids Signup and view all the answers

What is the primary application of DNA microarrays?

Gene expression analysis Signup and view all the answers

What is the temperature range for primer extension in PCR?

72°C Signup and view all the answers

What is the primary application of PCR in molecular biology?

Amplifying DNA fragments Signup and view all the answers

What is the primary purpose of comparative microarrays?

To compare gene expression in two different samples Signup and view all the answers

What does a yellow spot on a DNA microarray represent?

A gene that is expressed in both samples Signup and view all the answers

What is the purpose of labeling cDNA from sample 1 with green fluorescent dye?

To visualize the expression of genes in sample 1 Signup and view all the answers

What is the significance of a red spot on a DNA microarray?

The gene is only expressed in sample 2 Signup and view all the answers

What is the advantage of using comparative microarrays over other microarray techniques?

It provides a direct comparison of gene expression between two samples Signup and view all the answers

What is the relationship between relative fluorescent intensity and gene expression level?

Relative fluorescent intensity is directly proportional to gene expression level Signup and view all the answers

Study Notes

mRNA and cDNA

mRNA is produced when genes are transcribed, and 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 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

cDNA Cloning

cDNA cloning is used to make DNA copies of the transcribed mRNA
cDNAs are used as the introns are removed, and the clone size is smaller

DNA Sequencing

DNA sequencing allows us to determine the sequence of bases which can be used to predict the sequence of amino acids contained in the protein that the gene encodes
DNA sequencing provides full characterization of genes within the genome
DNA sequencing method devised by Sanger is the most common method (Nobel Prize 1980)
Automated DNA sequencing uses four ddNTPs, each labeled with a unique fluorescent marker
The DNA sequence fragments are separated on a capillary gel, and the fluorescence pattern produced shows the sequence of the DNA

Sequencing Technologies

Sequencing technologies have advanced, allowing the ability to sequence larger amounts of DNA quicker
Next-generation sequencing (NGS) uses sequential addition of nucleotides on microchips, short reads of 50-200bp, but very rapid, can sequence thousands to millions of DNA fragments per day

Genome Sequencing

In 2003, the human genome was completely sequenced, and we have sequence information for all genes in the genome
Current estimate is about 30,000 protein-coding genes

PCR

PCR (Polymerase Chain Reaction) is a method used to amplify DNA fragments
Amplified DNA fragments are separated by gel electrophoresis, and the PCR product size is equal to the amount of DNA between the primers
PCR can amplify DNA fragments from tens of bases in length to 5kb
Uses of PCR include amplifying whole genes or parts of genes from genomic DNA or cDNA for use in cloning and molecular analysis
PCR can amplify specific regions of DNA from a variety of samples, e.g., specific gene in different individuals to perform genetic diagnosis
PCR can amplify DNA from sources where DNA is limited, e.g., blood spots, fossils

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 is used in forensics to identify individuals from body fluids left at a crime scene or in paternity testing
PCR is used to check and confirm DNA constructs or transgenic animals

DNA Microarrays

DNA microarrays or gene chips are modern devices that use nucleic acid hybridization to rapidly measure which genes are expressed in a tissue sample
Comparative microarrays can be used to directly compare gene expression in two samples
Can be used to compare gene expression in normal and diseased cells

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Description

This quiz covers the process of converting mRNA to DNA through reverse transcription, and how it relates to gene expression in different cells. It also explores the importance of cDNA in expressing eukaryotic proteins and identifying genes expressed in specific cell types.