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1

List the 3 basic components required for a bacterial cloning vector and describe the purpose of each.

1. Origin of Replication:
ensures that the vector is replicated within the cells

2. Selectable markers: enable all cells containing the vector to be identified from those that don't contain the vector

3. Restriction sites: The point where DNA can be inserted into the vector. Needed so a DNA fragment can be inserted and replicated.

2

***** Look into slides

Describe, in order, the steps usually followed in producing recombinant DNA molecules in a plasmid vector.

1) The foreign DNA is cut by a restriction enzyme.

2) This fragment of DNA is then placed into the circular, bacterial DNA host (vector).

3) This plasmid that contains the foreign DNA is then ligated.

4) The plasmid is then able to replicate inside the bacterial host.


FROM THE SLIDES:

1. Plasmid vectors are isolated and cut with a restriction enzyme.
2. The DNA to be cloned is cut with the same restriction enzyme, producing a collection of fragments.
3. These DNA fragments are spliced into the vector and transferred to a bacterial host for replication.
4. Bacterial cells carrying plasmids with DNA inserts are identified by growth on selective medium and isolated.
5. The cloned DNA is then recovered from the bacterial host for further analysis.

3

Answer the following questions about standard PCR:

1. What enzyme is required for PCR? State the specific name of the enzyme, not just the type of enzyme.

2. Apart from the enzyme, what 3 DNA molecules are required (give the technical names by which they are called)? What must be true about the sequences of these molecules? Note that dNTPs are individual nucleotides, not DNA molecules.

3. Name the 3 basic steps of PCR and describe the molecular processes that occur in each. How is each step induced?

4. How does this system work to amplify DNA?

1. Taq Polymerase

2. Single DNA strand, and 2 primers that must be complementary to the single strands.

3.
Denaturization.
High temperatures make the DNA separate into single strands.

Primers Anneal.
Low temperatures allow complementary primers to anneal to the single DNA strands.

Elongation.
Taq polymerase picks up at the primers and finishes the process of creating a complementary strand to the DNA.
This step occurs at an intermediate temperature, comparative to the other temperatures.

4. Once the Taq Polymerase is finished, there is double the amount of DNA than there was at the start of the process. Thus the amount of DNA produced round after round increases exponentially.

4

One of the dominant features of the immune system is the capacity to generate new cells that contain different combinations of antibodies. Because there are billions of combinations it is impossible for each combination to be encoded by a single gene.
Explain in detail how such diversity is accomplished in the case of the light chain of a typical antibody.

Each mature B cell makes ONE type of light chain (kappa or lambda) and ONE type of heavy chain
An antigen stimulates a particular B cell with an antibody for that antigen
Produces population of plasma cells with antibody for that particular antigen.

The light chain of a typical antibody accomplishes diversity by being able to change based on any new threat.

5

expression vector

protein

6

What term is used to refer to the process in which DNA can be introduced into host cells.

Transformation

7

Of what advantage is it to have a polylinker region (Multiple unique restriction sites) embedded in the lacZ component in the pUC series of plasmids?

The lacZ gene is a selectable marker.
The colonies containing the newly inserted DNA will show up white because the LacZ gene will have been disrupted by the plasmid.
The normal colonies that don't contain the new DNA will be blue in the presence of the Xgal in the agarose plate.

8

BamHI cuts the sequence 5’ G|GATCC 3’.
Which of the following sequences would NOT be recognized by this enzyme?

3’ TCCTTAAG 5’

(choose the option containing no
G next to another G)

9

Which of the following statements about ddNTPs is true?

They have a hydrogen at the 3’ carbon of the ribose sugar.

10

2nd and 3rd generation, also called next generation sequencing approaches…

(select all that apply)

allow high throughput parallel sequencing

allow to sequence the entire transcript content of cells

use a wide range of different technologies

11

Nucleic acid blotting is widely used in recombinant DNA technology.
In a Southern blot one generally....

Hybridizes filter bound DNA with a DNA probe

12

A principal problem with inserting an unmodified mammalian gene into a bacterial plasmid, and then getting that gene expressed in bacteria, is

bacteria cannot remove Eukaryotic Introns

13

A human gene with a disease phenotype is going to be mapped by positional cloning.

Which would be most useful for this task?

Data about the inheritance of SNP markers in families with the disease.

14

Which of the following statements about manual Sanger sequencing is true?

The DNA sequence obtained is complementary to the template strand.

15

Assume that a plasmid (circular) is 3200 base pairs in length and has restriction sites at the following locations: 400, 700, 1400, 2600.

Give the expected sizes of the restriction fragments following complete digestion.

300, 700, 1000, 1200

16

When two proteins show a 50-70% match in amino acid sequence, it is likely…

The two proteins share a common ancestry

17

The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95°C.

Why would such a heat-stable polymerase be beneficial in PCR?

Each cycle includes a "hot" Denaturation phase (95°C), which Separates the Hydrogen Bonds that hold the strands of the template DNA together.

18

The human genome contains approximately 20,000 protein-coding genes, yet has the capacity to produce several hundred thousand gene products.
What can account for the vast difference in gene number and product number?

It is estimated that 40 to 60 percent of human genes produce more than one protein by alternative splicing.

19

Assume that one conducted a typical cloning experiment using pUC18, transformed an appropriate host bacterial strain (one carrying the lacZ complementing region), and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant pUC18?
Explain your answer.

The white would most likely contain the recombinant PU18.

20

This is the study of “all genes in an organism in their entirety.”

genomics

21

What is a concise definition of proteomics?

the process of defining the complete set of proteins encoded by a genome

22

In a microarray experiment, a researcher has noticed that the mRNA expression of the KC gene is elevated in cancer cells of the prostate, compared to control prostate cells.
The researcher initiates the analysis of the KC gene.
What is the technical term for this strategy of a genetic approach?


reverse genetics

23

A mutation has been recovered in a mutational screen that appears to strongly enhance the progression of prostate cancer in mice.
A researcher wants to determine the role of the gene in normal and pathological conditions.
What is the term for this general strategic approach?


Forward Genetics


(starts with phenotype and goes to gene) asked TA

24

In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling the second step?

The purpose of the initial high temperature is to denature the DNA strand; the cooling step is required for the primer to anneal--this lower temperature is optimal for a primer to anneal (and create a free 3’ hydroxy end in order to bind DNA polymerase.)

25

What appears to be the range of number of protein-coding genes per genome in eukaryotes?


5,000-45,000

26

In what way are specific DNA sequences of the template amplified in the polymerase chain reaction?
In other words, how does one target the target?

The sequences at the ends of the target DNA sequence must be known.
Then primers that are complementary to these end sequences are added to the PCR system and allowed to anneal to the DNA at these specific sites.
This must happen in order for Taq polymerase to begin DNA synthesis.
Thus DNA synthesis can be targeted to begin at the primers that you add into the system.

27

Restriction endonucleases are especially useful if they generate “sticky” ends.
What makes an end sticky?


Single-stranded complementary tails

28

What is the specific application of reverse transcriptase in the preparation of cDNA?

to take a RNA sequence and create a complementary DNA sequence from it

29

What is the function of dideoxynucleotides in Sanger DNA sequencing? (multiple choice)

a. They act as primers for DNA polymerase.

b. They act as primers for reverse transcriptase.

c. They cut the sequenced DNA at specific sites.

d. They allow only the specific sequencing of the RNAs of a genome.

e. They stop synthesis at a specific site, so the base at that site can be determined.


They stop synthesis at a specific site, so the base at that site can be determined.

30

Which technique would NOT be used to find a gene for a functional protein in a sequenced region of a genome?


See if a SNP database contains sequences in the region.