Nucleic Acids

Question 1

What is the difference between Type II and type I and III endonucleases?

Answer 1

Type II is the only type used in cloning

Question 2

T or F: recognition sequences are palindromic

Answer 2

True - they read the same from 3’ to 5’

Question 3

What is the consequence of type II restriction enzymes cutting closer to one end of the sequence?

Answer 3

• it creates an overhang ~4 bp in length (aka a sticky end)
• sticky ends allow for overlap so that fragments cut by the same restriction enzyme can be rejoined later to make a continuous double-stradned DNA sequence

NOTE: Blunt end cloning (cutting near the middle) also works but it is harder to join the blunt ends.

Question 4

T or F: sites for restriction enzyme cutting occur infrequently

Answer 4

False - 4 base pair sequences occur every ~256 base pairs, and 6 base pair sequences occur at every ~4000 base pairs

Question 5

What are EcoR1 and EcoRV?

Answer 5

The first and 5th restriction enzymes to be discovered, EcoR1 was derived from E. coli strand R

Question 6

What prevents bacteria from chopping up their own DNA?

Answer 6

They have a methylase enzyme that methylates the restriction binding site within their host cell. This prevents the restriction enzyme from binding and cleaving the site.

• New DNA (phage DNA) that enters the cell without methylation is cleaved at the commonly occuring restriction sites

Question 7

What two gel types are commonly used in gel electrophoresis?

Answer 7

Agrose or polyacrylamide

Question 8

If trying to perform gel electrophoresis on a DNA segment 20,000 bp in length what type of gel should be used?

Answer 8

Agrose.

The higher the concentration of the polymer, the smaller the holes in the gel and this can be adjusted for the size of DNA fragments that you wish to separate. Agarose gels are used to separate larger DNA fragments from a hundred bases to 10,000 -20,000 bases. Polyacrylamide is used for smaller DNA sizes from 20bp to about 700bp.

Question 9

How can the pores in agrose and polyacrylimide be made smaller?

Answer 9

By increasing the concentration

Question 10

What is mobility of DNA segments proportional to?

Answer 10

The inverse log of molecular weight (i.e Smaller fragments move faster through the pores of the gel and are found at the bottom of the gel). The approximate size of a fragment is determined by running a standard set of size markers on the same gel

mobility ~ 1/[log (MW)]

By making a semi-log plot of MW vs. migration distance in the gel, a straight line is obtained and the fragment size of an unknown length can be estimated from the graph.

Question 11

What are the basic step of the dideoxy chain termination method of DNA sequencing?

Answer 11

Aka Sanger Method

1. Reaction set up: Template DNA, DNA polymerase, and dNTPs are all present.
2. Primer that is complementary to the template strand is added and bind next to the site where religation happened via restriction enzymes (allows DNA polymerase to start making a new strand with dNTPs)
3. Primer extension makes DNA complementary to template strand
4. fluroescent terminators with no 3’ hydroxyl group are added in low concentration and DNA polymerase stops in the middle of synthesis when a terminator is encounter. (Different color terminator for A,C,T,G)
5. Special terminators (each base has its own terminator) without a 3-OH’ are added at low concentration causes termination at every possible length, each one ending in a different color dye
6. Capillary electophoresis separates out fragments by length and their color is read as they pass through from shortest to longest giving the full DNA sequence base-pair by base-pair

Question 12

What are three examples of next generation sequencing similar to that of dideoxy chain termination?

Answer 12

454 pyrosequencing
Illumina sequencing
semiconductor sequencing [post light sequencing]

NOTE: these increase throughput by eliminating electrophoresis

Question 13

Explain the steps of 454 pyrosequencing

Answer 13

1. single stranded DNA of different sizes with adaptors at each end attach to small beads in an oil-water emulsion so that adjacent DNA strands don’t touch
2. DNA on each bead is amplified using PCR so that millions of identical copies are attached to a single droplet (NO CLONING STEP)
3. Beads loaded onto a well plate with one bead per well and luciferase that is coupled to PPi (polyphosphate) release, lights up each time a base is added
4. As each base it added a CCD cameral records which beads are lighting up. This indicates the addition of whatever basepair was added in that cycle

Question 14

What are the read lengths and how many bases can be sequenced in a 10 hour 454 pyrosequencing run?

Answer 14

800 million bases (read length= ~800 bp)

Question 15

How does Illumina sequencing work?

Answer 15

1. adaptors are used to attach DNA to a solid transparent surface
2. Amplification gives tight clusters of about 1000 identical DNA fragments (~50 million of these clusters created)
3. each cluster measured by an optics system that uses lasers to excite clusters after a removable fluorescent dNTP has been added.
4. The cycle is then repeated

Question 16

How many base pairs of sequence and how many bases are generated with each run?

Answer 16

36 bp of sequence, 3 gigabases (equivalent to haploid human genome)

Question 17

Between the 454 pyrosequencing method and illumina method, which is better for assembling a genome with repeat sequences from scratch?

Answer 17

454 pyrosequencing, illumina is good for resequencing a known genome like the human genome

Question 18

How does ion sequencing [post-light sequencing] work?

Answer 18

1. like pyrosequencing the DNA is added to beads and amplified
2. beads are placed onto a chip and pH is measured. pH will drop if the correct base is added to the bead because a hydrogen is released each time a base is added by DNA polymerase. Since millions of identical copies of DNA are attached to each bead, when the correct nucleotide is added hydrogen ion concentration rises significantly.

Question 19

What are they advantages of the ion sequencing?

Answer 19

It is rapid and much more affordable

Question 20

How are microarrays created?

Answer 20

1. a silicon wafer is created and pieces of DNA (genes) are covalently attached to the surface
2. The location of each gene that has been placed on the wafer is known.
3. Two samples (control and experimental) of mRNA are reverse transcribed to cDNA and the control cDNA is dyed green and the experimental is dyed red
4. These red and green cDNA bind to make helical DNA with the complementary gene sequences on the surface of the wafer. The wafer is then washed
5. A laser is used to find areas of red, green, and yellow (red + green)

Question 21

What do areas of red, green, and yellow represent in a microarray?

Answer 21

Red - areas where mostly genes from the experimental sample are bound

Green - areas where mostly control genes are present

Yellow - areas where the gene is expressed in both the control and experimental sample

Question 22

What are some reasons to use microarrays?

Answer 22

To compare gene expression in two tissue types (cancer vs. normal)

To compare the change in a tissue type over time in response to a drug

Question 23

What is a heatmap?

Answer 23

Genes with the highest degree of expression pattern are grouped with algorithms

Ratios of red/green are taken at each gene with black indicating a 1:1 ratio of control to experimental.

Green - repressed areas (the gene has been downregulated compared to control)

Red - induced areas (the gene has been up regulated compared to control)

Genes with very similar expression patterns possibly work together in a single process like a pathway.

Question 24

What does PCR do?

Answer 24

Amplify small samples of DNA

Question 25

Why must the primers be used in the process of DNA replication in PCR?

Answer 25

heat is used to denature the protein and RNA primers must still be stable at this temperature to bind DNA

Question 26

What are the three steps of PCR?

Answer 26

1. denaturation- heat the DNA to separate (~94C @ 30 sec)
2. annealing primers
3. extension

Question 27

Why is DNA generated in cycle one of PCR longer than cycle II?

Answer 27

cycle I - DNA polymerase binds to primer and just keeps going to the end of the sequence

cycle II - the polymerase can only go until it runs off the side equal to the opposite primer

Question 28

What amplification is gained by repeating PCR twenty times?

Answer 28

1 million times

Question 29

How is it possible to add new amplified sequence in PCR?

Answer 29

• by synthesizing primers with 5’ extensions
• this is usually done to add restriction enzyme sites to the DNA so the PCR product can be cloned into a desired location

Question 30

What are type II restriction endonucleases, and what are they used for?

Answer 30

-The ability to identify, isolate and investigate a particular gene in this vast genome was virtually impossible until the discovery and commercial development of more than 100 bacterial type II restriction endonucleases.

They cut double stranded DNA at recognition sequences that are 4-8 base pairs in length and are palindromic (i.e. (both strands have the same sequence when read 5’ to 3’)

• They do not cut single stranded DNA or RNA
• Each enzyme cuts only specific recognition sequences

Question 31

How can PCR be used to identify human remains?

Answer 31

Humans contain regions of DNA that vary among individuals. These are called minisatellite DNAs or Variable Numbers of Tandem Repeats. PCR can be used to amplify these variable repeats (often CA repeats) and check the number of copies an individual has. Because we are diploid we usually have two different alleles with different numbers of repeats, so PCR gives two bands of different sizes on a gel. Some of these minisatellites are hypervariable with from 10-40 repeats. If enough of these are tested (6-10), no two people will be the same (except identical twins) and this is a foolproof way to identify a person or their remains.