Tools for Molecular Biology

Question 1

Palindrome

Answer 1

A sequence that reads the same after it has been flipped 180 degrees. They will read the same way from 5’ –> 3’

5’ GAATTC 3’
3’ CTTAAG 5’

Question 2

Restriction Enzymes

Answer 2

Cut double stranded DNA at specific nucleotide sequences. These sequences are often palindromic. They can produce cut ends that are flush (blunt) or that contain single nucleotide overhangs (sticky ends). Tend to recognize regions that are 4-8bp in length. They often work as dimers.

Question 3

Identify the types of DNA ends created by different restriction enzymes

Answer 3

sticky ends and blunt ends

Question 4

Determine the length of a DNA fragment from its electrophoreitic migration

Answer 4

The further it migrates, the smaller it is. We “run to the red” or to the anode (+) since DNA is negatively charged

Question 5

Recombinant DNA

Answer 5

“non-orginal” DNA. Those introduced from reannealing of DNA strands that weren’t originally together.

Question 6

Describe how a specific DNA molecule can be introduced into a plasmid for its subsequential propagation in a bacterial cell.

Answer 6

To create recombinant DNA in a plasmid, you start with the plasmid, cleave it with a restriction enzyme, add DNA fragments with the appropriate sticky ends that you want to be cloned that will then join in the plasmid with ATP and ligase to seal the ends. The plasmid can then be transformed into a bacterial cell which will replicate it. When you want the DNA then, you extract the plasmids and use the same restriction enzymes to cut it out.

Question 7

Explain how recombinant DNA can be used in medicine.

Answer 7

Some recombinant plasmids can have the regulatory elements to create proteins. Such an example is generating insulin. If we put coding sequence for insulin into a plasmid and transform it into bacteria, the bacteria will create the protein which can then later be isolated.

Question 8

When to use a polyacrylamide gel

Answer 8

when DNA is less than 200bp

Question 9

When to use a pulse field

Answer 9

For large DNA greater than 50,000bp. Here the electric field is shifting periodically.

Question 10

What is hybridization?

Answer 10

It is bringing two single-stranded pieces of DNA together. It is renaturation of DNA. This is the basis for southern blot analysis.

Question 11

Describe the principles behind southern blotting and northern blotting

Answer 11

Southern blotting combines the techniques of electrophoresis and hybridization to detect specific DNA molecules. Northern blotting is very similar but it is used to detect RNA. You use them to detect a specific DNA or RNA molecule in a complex mixture by gel hybridization techniques. It is using a labeled probe as well.

Question 12

Probes

Answer 12

single stranded DNA molecules used to detect complementary sequences. They often carry radioactive or chemical markers to facilitate their detection. You can also get imperfect base pairing if you lower the condition.

Question 13

Explain the term restriction fragment length polymorphism (RFLP) when comparing DNA from two different individuals.

Answer 13

It is the concept of removing a restriction site which thus alters the fragment lengths generated when treated with the restriction enzyme. If you are looking at an individual with sickle-cell for example, the MstII restriction enzyme cut site is destroyed, thus, when you compare the DNA of that individual with someone that is normal, they will have a longer fragment on a southern blot treated with a probe.

Question 14

Discuss how in situ hybridization might be used in diagnosis of disease.

Answer 14

In this technique, cells can be isolated and heated to create single stranded DNA. This is then treated with a fluorescent probe that can hybridize with the single strand that it is complementary to and be visible. In the diagnosis of disease, one can probe for a specific gene that is known to cause that disease, such as increased levels of Her2 in some cases of breast cancer. If the gene is more prevalent, you can accurately indicate someone is at risk.

Question 15

Polymerase Chain Reaction (PCR)

Answer 15

It is a powerful way to amplify a specific DNA sequence. The power of PCR lies in the selectivity of DNA hybridization of the primers to a specific sequence and the ability of DNA polymerase to faithfully copy DNA through repeated rounds of synthesis in vitro. Billions of copies can be obtained in just a few hours due to it copying exponentially. You mix primers, DNA, nucleotides, polymerase and a buffer solution. The primers are designed to be before and after the gene of interest. Involves ANNEALING, EXTENSION, DENATURATION. It uses two primers that are specific for the 3’ end and thus bind to each strand. In a clinical setting, it is useful to amplify DNA from a limited sample to then test.

Question 16

Multiplex PCR

Answer 16

Can be used to run a PCR reaction using multiple sites for amplification. ALL PCR PRODUCTS HAVE TO BE DIFFERENT SIZES IN ORDER TO SHOW UP ON A GEL THOUGH. If there is a deletion of a region of the exon, the primer cannot bind to it and amplify it.

Question 17

Describe how cDNA is prepared from RNA

Answer 17

It utilizes Reverse Transcriptase to create complementary DNA or cDNA that can be used in PCR to analyze RNA viral infections or to determine what genes are being transcribed in a cell. First, you isolate out RNA, then since RNA has a poly-A tail, you can create a primer that is specific to that region. The transcriptase then copies the RNA to DNA. RNAse H then degrades the RNA and then synthesize the second strand of DNA using polymerase and the RNA acts as a primer.

Question 18

Microarray Analysis

Answer 18

They are used to monitor the expression of thousands of genes simultaneously and are based on hybridization techniques. Can measure many genes at the same time. It allows us to look at the transcriptome. It starts with a tissue sample that you isolate the mRNA from. The mRNA is reverse transcribed to cDNA. The cDNA is fluorescently labeled and applied to a glass slide (microarray) with complementary DNA attached to it that hybridizes with the cDNA of interest. The color represents that the gene is expressed. By looking at genes for cancer for instance, depending on the genes present, certain treatment options might be more beneficial.

Question 19

Traditional Sequencing

Answer 19

It is similar to PCR except that only ONE primer is used and the reaction is “spiked” with fluorescently labeled dideoxyribonucleotides. These are readily incorporated into the a growing nucleotide chain, but because they have no reactive 3’-hydroxyl, they stop the chain growth because there is not another spot to add the next nucleotide. Over all of the reactions, one of these terminators will be added at every location. Then the results are ran on a polyacrylamide gel to separate by size and the labeled terminators are identified at each location.

Question 20

Next Generation Sequencing (NGS)

Answer 20

It is a “catch-all” phrase referring to several different technologies that allow sequencing of millions of short DNA fragments simultaneously. NGS is preferred for large scale genome analysis because it is cheaper, faster, requires less DNA, and is more accurate than traditional sequencing. Illumina is a type of NGS that is also based on the dideoxy logic. Each nucleotide is attached to a removable fluorescent molecule as well as a special chain terminating chemical adduct that can also be removed chemically. Sequencing is carried out, one of these nucleotides is added, blocks elongation, takes a photo, then removes the block and fluorescent region and continues the process. A computer will then determine the overall sequence via overlap. These are compared to a reference sequence to determine errors. You get the same nucleotide ran several times so it is more accurate b/c we have depth of coverage.

Question 21

NGS vs. Traditional Sequencing

Answer 21

NGS is:

1) cheaper- requires more investment initially but will pay off in the end.
2) requires less DNA
3) more accurate
4) Faster