Lecture 16 - Nucleic Acids and their Detection

Question 1

What does looking at nucleic acids using biological methods allow you to do?

Answer 1

It allows you to make qualitative and quantitative analyses of them.

Question 2

Which type of analysis is about the sizes, nucleotide composition (sequence changes), conformational/configuration changes that we can detect, and structures?

Answer 2

Qualitative Analysis

Question 3

Why is quantative analysis of nucleic acids important?

Answer 3

Quantitative analysis of nucleic acids is important because very often, level of gene product have very important consequences in physiology and health. Measuring the various levels of a specific substance can allow us to diagnose certain diseases or to give some prognosis. In a clinical lab, for example, we can assess oncogene products, tumour markers, and tumour suppressors. It is also important to help understand the variances that might exist in a certain population.

Question 4

What principle do nucleic acid detectors rely on to function?

Answer 4

Complementary Base-Pairing

Question 5

Why are probes used?

Answer 5

Often, you can never see the little bits of DNA or RNA that you are studying. But, if you attach a large probe to the strand (via complementary pairing), you can visualize them.

Question 6

Describe the process of molecular probing.

Answer 6

• A complex mixture is seperated via agrose gel electrophoresis.
• The nucleic acids are transported to a solid phase support (typically some kind of membrane) so that their positions are maintained.
• A complementary probe is labelled and mixed with the membrane.
• All probes that didn’t interact with a protein are washed away.
• The probes are visualized (based on type of probe, radioactive probe are visualized with x-ray film for example) so that you can see the regions on the membrane that correspond to the sequence.

Question 7

Which enzyme is used to label single-stranded oligonucleotides?

Answer 7

Polynucleotides Kinase

Question 8

What is polynucleotides kinase (PNK)?

Answer 8

PNK is a bacteriophage enzyme used to label single-stranded oligonucleotides.

Question 9

How does PNK label oligonucleotides?

Answer 9

PNK attaches a radio-labelled phosphate, the gamma phosphate (the final phosphate), from an ATP molecule to the 5’ end of the oligonucleotide.

Question 10

What are you left with after PNK labels an oligonucleotide? How do you handle this?

Answer 10

After labelling, you are left with radioactive ADP and a oligonucleotide where the 5’ end is labelled with a radioactive phosphate. You can get rid of the unused ATP and ADP via liquid chromatography. The oligonucleotide will come down the column faster than the ATP and ADP which will get caught in the pores of the beads.

Question 11

How is PCR used to label proteins?

Answer 11

You can make probes by incorporating labelled nucleotides into an elongating strand of DNA. You can do this by carring out PCR with isotropic radiolaelled nucleoside triphosphates.

Question 12

Describe the processing of making labelled DNA probes via PCR.

Answer 12

• Denature the strands of DNA and add the primers.
• Reduce the concentration of dCTP and add α-³²P dCTP. (α because its most proximal to the 5’ end and will be incorporated into the growing DNA chain..)
• Polymerase incorporates the nucleotides, including the radiolabelled cytosines.
• The unincorporated radio-dNTPs are removed via gel-filtration chromatography.
• The strand is boiled to denature it into a single-stranded DNA.
• The strand is introduced to its target.
• Loosely bound markers (not exact matches) are removed by washing them off or increasing temperature.

Question 13

Since DNA is giant, what do you do to it before you analyze it (perform agarose gel electrophoresis)?

Answer 13

You cut it into pieces using a restriction enzyme.

Question 14

Describe the process of transferring nucleic acids to nylon or nitrocellulose membranes.

Answer 14

• DNA is cut by restriction enzymes and then run through agarose gel electrophoresis.
• The strands in the gel are denatured in a bath of NaOH.
• The agarose gel is placed on top of a bath of alkaline solution containing NaOH.
• The solid phase support membrane (usually nylon-based or nitrocellulose since they are positive enough to hold the DNA in place) is placed on top of the agarose gel.
• A large amount of filterp paper around the gel and membrane absorbs the liquid, pulling up water to bring the DNA from the gel to the membrane.
• UV light is used to cross-link the DNA to the nylon or nitrocellulose by covalent bonds, creating a permanent record.

Question 15

What does hybridizing a membrane of DNA do?

Answer 15

Hybridization is when you introduce a probe to a membrane containing DNA. You are forming a hybrid between the probe and the DNA target.

Question 16

What is the product of hybridization called?

Answer 16

Southern Blot

Question 17

What should you do to a radio-marked Southern blot to determine the quantity and position of desired DNA present?

Answer 17

You subject the membrane to x-ray film and then examine where the radioactivity exposed the silver grains on the film. You can then calculate the number of moles present in a given sample by considering the amount of probes you used and how much DNA you loaded.

Question 18

What are the benefits to performing a Southern blot? What do you need to know going into it?

Answer 18

You can use Southern blots to pedigree individuals or orgnisms with very little understanding of sequence information. This method can be done efficiently and cheaply based on the idea that you have a probe that corresponds to a specific region of interest and by understanding how these particular DNA molecules behave when cut by a given enzyme.

Question 19

What would the difference be between the two following Southern blots?:

1. A restriction enzyme cuts in the middle of the gene corresponding to the probe.
2. A polymorphism changes a single nucleotide in the gene corresponding to the probe such that the restriction enzyme no longer identifies the restriction site.

What does this tell us about variation detection?

Answer 19

Scenario 1 would result in a Southern blot with two bands, corresponding to each half of the cut gene (since the probe can bind to both). Scenario 2 would result in a Southern blot with only a single band.

You can detect a variation in the sequence without knowing much about the sequence except that there is a polymorphism thanks to the molecular probe.

Question 20

What do multiple bands in a Southern blot indicate?

Answer 20

Multiple bands indicate that there are multiple nucleotides sizes. This indicates that some genes were cut by restriction enzymes while others were not.

Question 21

What technique can be used to detect polymorphisms without using a Southern blot?

Answer 21

PCR

Question 22

What kind of blot is analyzes RNA?

Answer 22

Northern Blot

Question 23

Describe the process of a Northern blot.

Answer 23

• RNA is denatured (it will twist otherwise) by heating them up and placing them on a layer of a denaturing agent like formaldehyde.
• They are separated by size in an agarose gel (with formaldehyde still present to keep them denatured).
• The RNA is transferred to a nitrocellulose or nylon membrane to keep them in their relative positions.

Question 24

What is dehybridizing?

Answer 24

Dehybridizing is when you wash all the probes off a Northern or Southern blot so that they can be reused.

Question 25

What can Northern blots be used to identify in humans?

Answer 25

Humans have stage-specific or temporally controlled mRNA expressions as well as organ specific expression of mRNAs in the body that can be identified via Northern blots (by comparing young person to old person or by comparing two different organs).

Question 26

Do Northern blots obtain quantitative or qualitative information? Explain.

Answer 26

Both!

They are qualitative because if the probe may sometimes recognize differing targets, telling you if there is a variant due to alternative splicing or another reason.

They are quantitative because you can see how much of a RNA is present.