ignore - Optional Tutorial 1: Molecular Tools

Question 1

In gel electrophoresis i) __________ DNA fragments travel faster so they appear closer to the ii) ___________ of the gel after samples are run.

A i) larger; ii) top

B i) smaller; ii) bottom

C i) larger; ii) bottom

D i) smaller; ii) top

Answer 1

B - The agarose polymer creates a mesh-like network in the agarose gel that impedes the movement of larger molecules so that smaller DNA fragments travel faster and further along the gel.

Question 2

Imagine you have four samples of DNA labeled, A, B, C, and D. You perform a Southern Blot to determine which of these four samples contains a specific DNA sequence of interest. You run these samples side by side in separate lanes on the electrophoresis gel, transfer to a nitrocellulose membrane and incubate them with a probe for the sequence of interest. If only samples B and D contain the sequence of interest what will your final autoradiogram look like?

Answer 2

Because only samples B and D contain the sequence of interest, the probe will only bind the DNA in these lanes. DNA is present in all lanes but will only appear on the autoradiograph where the probe is bound. Therefore the autoradiogram will have a band in lanes B and D only.

Question 3

Now imagine you run this experiment again but this time you are testing for two new sequences of interest, sequence 2 and sequence 3. You run these samples again on two electrophoresis gels, transfer and incubate each membrane with a separate probe; Probe 2 and Probe 3, complimentary to these two sequences of interest. If only samples A and C contain sequence 2, and only sample D contains sequence 3, what will your autoradiograms look like?

Answer 3

will only see bands in the A and C lane for probe 2 and lane D for probe 3 as these are the only probed sequences of interest

Question 4

If the gene locus encoding the IgM heavy chain variable domain in mice has 90 V, 25 D, and 6 J segments, and the Lambda light chain variable domain has 75 V and 10 D segments, how many possible combinations exist for the variable region of the IgM/Lamda Ab?

Answer 4

= Heavy x light
= (V x D x J)(V x D)
= (90 x 25 x 6)(75 x 10)
= 10 125 000 combinations

Question 5

If gene locus encoding the IgG heavy chain variable domain in rabbits has 60 V, 32 D, and 8 J segments, and the Kappa light chain variable domain has 86 V and 11 D segments, how many possible combinations for the variable region of the IgG/Kappa Ab are there?

Answer 5

= Heavy x light
= (V x D x J)(V x D)
= (60 x 32 x 8)(86 x 11)
= 14 530 560 combinations

Question 6

Fill in the blanks: A mouse anti-GAPDH antibody is a ________ (primary/secondary) antibody that recognizes the antigen ________.

Answer 6

primary, GAPDH

Question 7

Fill in the blanks: A goat anti-mouse antibody is a ________ (primary/secondary) antibody produced in ________.

Answer 7

secondary, goats

Question 8

What is the name of the gel used in gel electrophoresis?

Answer 8

agarose gel

Question 9

What is the purpose of gel electrophoresis?

Answer 9

to separate molecules via their size and charge

Question 10

What does hybridization mean?

Answer 10

the combination of 2 complementary DNA strands that originate from different sources

Question 11

what do dsDNA and ssDNA stand for?

Answer 11

double-stranded and single-stranded

Question 12

What is the purpose of a southern Blot?

Answer 12

it is a blotting system to find a specific DNA sequence from a larger sample of DNA

Question 13

What is the difference between the southern and northern blot?

Answer 13

southern detects a specific DNA sequence while northern detects a specific RNA sequence

Question 14

what is an autoradiogram?

Answer 14

a diagram that only shows hybridized bands of DNA/RNA.

Question 15

What is the purpose of Fluorescent in-situ hybridization?

Answer 15

it hybridizes whole chromosomes therefore it is used to produce karyotypes

Question 16

What is the purpose of RNA in-situ hybridization?

Answer 16

this hybridizes mRNA transcript sequences allowing for the analysis of gene expression

Question 17

What is a
a) restriction enzyme
b) restriction site

Answer 17

a) group of endonuclease that cleaves foreign DNA
b) site in which restriction enzymes cleave

Question 18

most restriction sites are 4 to 8 Bp long and are considered palindromic. What does this mean?

Answer 18

when read 5’ to 3’ both strands have the same sequence

Question 19

What is the difference between a blunt end and a sticky end? Which is more common?

Answer 19

blunt end is when the restriction enzymes cleave straight through the 2 complementary strands
sicky end is when the restriction enzyme cleaves both strands at different locations resulting in an overhang from both strands.

Question 20

What are the 4 steps to DNA cloning?

Answer 20

1. digestion = cleave plasmid at MCS and destroy DNA sample into fragments
2. ligation = due to the sicky end of the plasmid being complementary to the denatured DNA and the presence of DNA Ligase the target fragment is inserted in the DNA
3. transformation = mix the recombined plasmid w/ cells in order to encourage the cells to take in the recombinant plasmid
4. selection = place the cells on a medium that contains a specific set of antibodies which will kill off any cells that do not contain the recombined plasmid

Question 21

describe clone screening

Answer 21

the act of using gel electrophoresis to identify the recombinant plasmids from the non-recombinant plasmids

Question 22

What basic form do all antibodies (Abs) come in?

Answer 22

a protein with 4 polypeptides in which 2 are heavy chains and 2 are light chains

Question 23

what are the 3 domains of Ab’s? Which one accounts the most for the high specificity of Ab’s

Answer 23

1) constant
2) variable
3) hyper-variable - contains the most diversity

Question 24

it is said that the antigen binding site binds to the epitope b/w an Ab and Ag (antigen) complex. Which site belongs to which molecule?

Answer 24

antigen binding site is on the Ab while the epitope is on the Ag

Question 25

a combination of non-covalent forces is what strengthens the binding of the Ab-Ag complex. T or F?

Answer 25

T

Question 26

What are the 5 classses of heavy chains in Ab’s

Answer 26

IgA, IgD, IgE, IgG, IgM

Question 27

What are the 2 classes of light chains in Ab’s

Answer 27

lambda and kappa

Question 28

what is the main reason for the specificity of Ab’s in the variable region of Ab’s

Answer 28

the chromosomal rearrangement that occurs during the development of B cells which produce Ab’s

Question 29

What are the 2 types of Ab’s?

Answer 29

primary = targets a specific protein of the same species
secondary = originates from a different species and binds to the primary Ab of that species while targeting specific proteins

Question 30

What are the 2 techniques using an antibody-based detection sys?

Answer 30

1) immunofluorescence = used to locate a visualize a specific protein
2) immunoprecipitation = used to pull out a specific target protein