MCBG Session 14 - Mutagenesis

Question 1

Compare and contrast how DNA is analysed at a nucleotide, gene and chromosome level.

Answer 1

• Analysis of DNA at nucleotide level

I. DNA sequencing

II. PCR plus restriction analysis

• Analysis of DNA at gene level

I. Southern hybridisation

II. Northern hybridisation

III. RT-PCR

IV. Microarray

V. DNA fingerprinting/ DNA profiling

• Analysis of DNA at the chromosome level

I. Karyotyping

II. FISH/Chromosome painting

Question 2

Discuss the ethics behind DNA sequencing.

Answer 2

• Who would be interested in your genome information?

I. Family

II. Potential spouse

III. Doctor

IV. Government

V. Police

VI. Schools

VII. Insurance companies

• Can the knowledge help prevent illness later in life?
• Does it open up areas for discrimination?
• Who owns DNA sequence?

Question 3

What is RT-PCR?

Answer 3

• Reverse transcription polymerase chain reaction (RT-PCR), a variant of polymerase chain reaction (PCR), is a technique commonly used in molecular biology to detect RNA expression.
• Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different.
• Traditional PCR is used to exponentially amplify target DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase.
• Subsequently, the newly synthesized cDNA is amplified using traditional PCR.

Question 4

Outline the principles and practice of DNA fingerprinting.

Answer 4

• DNA profiling (also called DNA fingerprinting, DNA testing, or DNA typing) is a forensic technique used to identify individuals by characteristics of their DNA.
• A DNA profile is a small set of DNA variations that is very likely to be different in all unrelated individuals, thereby being as unique to individuals as are fingerprints (hence the alternate name for the technique).
• DNA profiling is used in, for example, parentage testing and criminal investigation, to identify a person or to place a person at a crime scene, techniques which are now employed globally in forensic science to facilitate police detective work and help clarify paternity and immigration disputes.
• Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different that it is possible to distinguish one individual from another, unless they are monozygotic (“identical”) twins.
• DNA profiling uses repetitive (“repeat”) sequences that are highly variable, called variable number tandem repeats (VNTRs),
• in particular short tandem repeats (STRs). VNTR loci are very similar between closely related humans, but are so variable that unrelated individuals are extremely unlikely to have the same VNTRs.

Question 5

Outline the procedure for creating a DNA fingerprint.

Answer 5

• The DNA is extracted from the sample cells (e.g. skin, hair, or blood cells) and purified.
• In Jeffreys’ original approach, which was based on restriction fragment length polymorphism(RFLP) technology, the DNA was then cut at specific points along the strand with proteins known as restriction enzymes.
• The enzymes produced fragments of varying lengths that were sorted by placing them on a gel and then subjecting the gel to an electric current (gel electrophoresis): the shorter the fragment, the more quickly it moved toward the positive pole (anode).
• The sorted double-stranded DNA fragments were then subjected to a blotting technique in which they were split into single strands and transferred to a nylon sheet.
• The fragments underwent autoradiography in which they were exposed to DNA probes—pieces of synthetic DNA that were made radioactive and that bound to the mini-satellites.
• A piece of X-ray film was then exposed to the fragments, and a dark mark was produced at any point where a radioactive probe had become attached.
• The resultant pattern of marks could then be analysed.

Question 6

What is FISH?

Answer 6

• Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.
• It was developed by biomedical researchers in the early 1980s and is used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
• Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes.
• FISH is often used for finding specific features in DNA for use in genetic counselling, medicine, and species identification.
• FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumour cells, and tissue samples
• . In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
• FISH is used by examining the cellular reproduction cycle, specifically interphase of the nuclei for any chromosomal abnormalities