Week 10 Gene Isolation and Manipulation

Question 1

What types of blotting is used for DNA, RNA, and proteins respectively

Answer 1

Southern, Northern, Western

Question 2

What does gel eletrophoresis do?

Answer 2

It separates mixtures of DNA fragments, RNAs or proteins based on physical properties such as size and charge
Gel is the matrix used t separate molecules and electrophoresis is the voltage that is applied
DNA and RNA have overall negative charges so will migrate from negative to positive depending on their size

Question 3

What are the steps that follow gel electrophoresis?

Answer 3

Transfer to a membrane
Addition of the probe

Question 4

What are the probes in Southern and Northern blotting?

Answer 4

Radioactive single stranded nucleic acids that are complementary to the nucleic strand of interest
- will hybridize to the complementary strand
-unbound probe is washed away and hybridization is revealed by autoradiography (x-ray)

Question 5

Restriction enzymes are ___ on opposite strands

Answer 5

Palindromic

Question 6

Why does cutting chromosome sized DNA occur

Answer 6

Since it is do big, it is easier to work with and be analyzed in smaller fragments

Question 7

____ cut DNA at specific
sequences, producing
fragments with staggered
or blunt ends.

Answer 7

Restriction enzymes

Question 8

What is qPCR?

Answer 8

Quantifying PCR, measures the amount of DNA product in “real
time” during each PCR cycle.
-measures the intensity of a fluorescent signal

Question 9

DNA cloning

Answer 9

a method for isolating a particular sequence of DNA from a complex mixture of different DNA sequences - needs to be inserted into a vector

Question 10

Force directional cloning

Answer 10

Solves problem where the gene could orient itself either way into the vector/the vector could just reanneal
- force directional cloning uses two different restriction enzymes to make sticky ends not complementary to each other, will have compatible ends on only one side each, when they come together and when they anneal they can only go one way
Also means that the plasmid can’t just reanneal to itself

Question 11

Practical features of plasmid vectors

Answer 11

Polylinkers
Origin of Replication
Drug Resistance Gene

Question 12

Genomic library vs cDNA library

Answer 12

Gene libraries represent all of the genes in an organisms whereas cDNA libraries represent only those genes that were expressed in the cell that were a source of mRNA

Question 13

How to identify a clone of interest from a genomic of cDNA library

Answer 13

Colonies on a petri dish are transferred to a membrane
Lyse bacterial cells and denature DNA
Add probe
Autoradiograph the locate the desired clone
Pick up positive clone
-Grow and amplify the colony

Question 14

Cloning by PCR

Answer 14

Use PCR primers with
restriction sites at their 5′
end to produce PCR
products with sticky ends

Can also use Gibson Assembly (multiple linear DNA fragments that have sequence similarity at their ends)

Question 15

What is the most common way to sequence DNA?

Answer 15

Dideoxy sequencing/Sanger sequencing

Question 16

Transgenes are introduced into eukaryotic cells by chemical, physical, and biological methods such as:

Answer 16

-Chemical: lipid vesicle being engulphed
Physical: electroporation, biolistic particle delivery, microinjection
Biological: bacteria or viruses

Question 17

Genetic engineering in yeast

Answer 17

Transgenic yeast cells are generated by homologous recombination between a yeast chromosome and a plasmid that is transformed into yeast and carries a gene of interest

Question 18

Genetic engineering in plants

Answer 18

Transgenic plants are generated by random insertion into a chromosome of a Ti plasmid that carried a gene of interest and is delivered by Agrobacterium tumefaciens

Ti plasmid causes an infected plant to produce uncontrolled growths called tumors or galls
-Scientists are able to eliminate all of the T-DNA sequence between the borders (including the tumour causing gene) and replace it with a gene of interest and a selectable marker

Question 19

Transgenesis in C. elegans

Answer 19

Transgenic worms are generated by injection of a plasmid
containing a gene of interest into the gonad.

Question 20

Gene knockout

Answer 20

An inactive gene is targeted to replace the functioning gene in a
culture of ES cells (embryonic stem cells)

Question 21

The three stages of gene knockout

Answer 21

An inactive gene is targeted to replace the functioning gene in a culture of ES cells, producing ES cells containing a gene knockout

ES cells containing the inactive gene are transferred to mice embryos
(Targeted insertion of transgene by homologous recombination)

Knockout mice are identified and bred to produce mice of a known genotype (selection of cells with gene knockout)