PCR and Molecular Biology

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Questions and Answers

What is the primary function of the enzyme DNA polymerase in PCR?

To amplify RNA molecules

To synthesize new DNA strands (correct)

To ligate DNA fragments

To degrade DNA strands

What is the purpose of oligonucleotides in PCR?

To inhibit DNA synthesis

To ligate DNA fragments

To serve as primers for DNA synthesis (correct)

To degrade RNA molecules

What is the main difference between PCR and RT-PCR?

PCR amplifies DNA, while RT-PCR amplifies RNA

PCR is used for RNA, while RT-PCR is used for DNA

PCR is used for genomic DNA, while RT-PCR is used for cDNA

PCR uses DNA polymerase, while RT-PCR uses reverse transcriptase (correct)

What is the purpose of the second primer in RT-PCR?

To convert the single-stranded DNA to double-stranded DNA Signup and view all the answers

What is the result of using an oligo (dT) primer in RT-PCR?

Synthesis of a cDNA complementary to all mRNAs Signup and view all the answers

What is the purpose of heat-stable Taq DNA polymerase in RT-PCR?

To amplify the double-stranded DNA Signup and view all the answers

How are the products of RT-PCR amplification typically analyzed?

By gel electrophoresis Signup and view all the answers

What is the meaning of the Greek word 'klon' in the context of gene cloning?

twig Signup and view all the answers

Who performed the first cloning experiment in 1973?

Stanley Cohen and Herbert Boyer Signup and view all the answers

In what year was human growth hormone (hGH) produced in E. coli?

1985 Signup and view all the answers

Who discovered restriction endonucleases in E. coli?

Stewart Linn and Werner Arber Signup and view all the answers

What is the result of a cloning experiment?

A group of identical cells Signup and view all the answers

In what year was human insulin, the first recombinant DNA product, approved?

1982 Signup and view all the answers

Who cloned a sheep named Dolly in Scotland in 1997?

Not specified in the text Signup and view all the answers

What is the primary application of RT-PCR?

To identify the presence of a specific gene Signup and view all the answers

What is the purpose of the reporter probe in real-time PCR?

To release a fluorescent tag when the target DNA is amplified Signup and view all the answers

What is the advantage of real-time PCR over traditional PCR?

It provides quantitative data on the amplification of target DNA Signup and view all the answers

What is the benefit of using restriction site sequences on the PCR primers?

It allows for the ligation of the cDNA into a vector Signup and view all the answers

What is the outcome of using RT-PCR to analyze a sample?

A qualitative measurement of the presence of a specific gene Signup and view all the answers

What is the role of the fluorescent tag in real-time PCR?

It is released when the target DNA is amplified Signup and view all the answers

What is the significance of the quenching tag in real-time PCR?

It reduces the fluorescence signal Signup and view all the answers

What is the advantage of using M13 phage vectors?

Single-stranded DNA can be recovered in single-stranded form. Signup and view all the answers

What is the purpose of Fluorescence In Situ Hybridization (FISH)?

To examine the presence or absence of a particular DNA sequence. Signup and view all the answers

What is the result of C banding in chromosome identification?

Staining the centromeric region of each chromosome. Signup and view all the answers

What is the purpose of Comparative Genome Hybridization (CGH)?

To provide a high-resolution assessment of the genome. Signup and view all the answers

What is the characteristic of cosmids?

They are designed especially for cloning large DNA fragments. Signup and view all the answers

What is the purpose of electroporation?

To drive the DNA into cells. Signup and view all the answers

What is the characteristic of Charon phages?

They can accept up to 20 kb of DNA. Signup and view all the answers

What is the result of high-resolution banding in chromosome identification?

The resulting dark and light bands are the reverse of those produced by G or Q banding. Signup and view all the answers

What is the purpose of phagemids?

To produce single-stranded DNA. Signup and view all the answers

Study Notes

PCR (Polymerase Chain Reaction)

Amplifies a region of DNA between two predetermined sites using oligonucleotides as primers
Each cycle of PCR doubles the number of copies of the amplified DNA until a large quantity is made
Developed by Kary Mullis and his colleagues in the 1980s

Reverse Transcription-PCR (RT-PCR)

Uses the enzyme reverse transcriptase to synthesize DNA strands that are complementary to RNA templates
Can be used to clone a cDNA from just one mRNA
Starts with an mRNA and reverse transcribes it to make a single-stranded DNA, then uses a forward primer to convert it to double-stranded DNA
The resulting DNA strands can be amplified by standard PCR

Analysis of RNA by RT-PCR

Uses RT-PCR to generate a cDNA from a single type of mRNA
The sequence of the mRNA must be known to design the primer for the PCR step
Restriction site sequences can be placed on the PCR primers, making it easy to cleave and ligate the cDNA into a vector
Many modifications of the RT-PCR procedure have been developed to make it more quantitative
Allows for the quick and easy determination of whether a particular gene is being transcribed

Real-Time PCR

Quantifies the amplification of a DNA as it occurs (in real-time)
Uses a reporter probe that is annealed to the DNA strand, which has a fluorescent tag and a fluorescence quenching tag
As the DNA polymerase extends the primers, the reporter probe is degraded, separating the fluorescent tag from the quenching tag, allowing for the measurement of fluorescence

Gene Cloning

A product of any cloning experiment is a clone, a group of identical cells or organisms
The first gene cloning experiment was performed by Stanley Cohen, Herbert Boyer, and their colleagues in 1973
Clones of identical frogs were created by John Gurdon, and the first mammal to be cloned was a sheep named Dolly in 1997

The Role of Restriction Endonucleases

Discovered by Stewart Linn and Werner Arber
Used in gene cloning to cut DNA at specific sequences

Vectors

Phages as vectors have a natural advantage over plasmids
Charon phages can accept up to 20 KB of DNA
Cosmids are designed for cloning large DNA fragments and can behave as both plasmids and phages
M13 phage vectors produce single-stranded DNA, which can be recovered in single-stranded form
Phagemids produce single-stranded DNA and have characteristics of both phages and plasmids

Chromosome Identification

There are three commonly used staining methods to distinguish among human chromosomes: G-banding, Q-banding, and R-banding
G-banding is the most common method used in clinical laboratories
Q-banding requires staining with quinacrine mustard or related compounds and examination by fluorescence microscopy
R-banding involves heating before staining, resulting in dark and light bands that are the reverse of those produced by G or Q banding

Special Cytological Procedures

C-banding stains the centromeric region of each chromosome and other regions containing constitutive heterochromatin
High-resolution banding (prometaphase banding) is achieved through G-banding or R-banding techniques to stain chromosomes
Fragile sites are non-staining gaps that are occasionally observed at characteristic sites on several chromosomes

Fluorescence In Situ Hybridization (FISH)

A technique to examine the presence or absence of a particular DNA sequence or to evaluate the number or organization of a chromosome or chromosomal region
Identifies particular chromosomal rearrangements or rapidly diagnoses the existence of an abnormal chromosome number in clinical material

Chromosome and Genome Analysis by Microarrays

Comparative genome hybridization (CGH) complements conventional karyotyping and provides a more sensitive, high-resolution assessment of the genome
Reveals variants, particularly small changes in copy number between samples, that are of uncertain clinical significance

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Description

Learn about PCR, real-time PCR, nucleotide sequences of genes and chromosomes, and their principles from chapters 14 of 'Principles of Genetics' and 4 of 'Molecular Biology'.