Oct 2 DNA Cloning

Question 1

what is recombinant DNA technology?

Answer 1

vector + DNA fragment
-> recombinant DNA
-> replication of recombinant DNA within host cells
-> isolation, sequencing, and manipulation of purified DNA fragment

Question 2

what are plasmids and how are they related to DNA cloning?

Answer 2

most common vector used in recombinant DNA technologies
circular
double stranded DNA (dsDNA)
extrachromosomal
found in bacteria and lower eukaryotes
replication occurs before cell division

Question 3

what do restriction endonucleases/enzymes do?

Answer 3

cleave, or cut phosphodiester bonds usually in a symmetrical fashion

Question 4

what are the different libraries and what can be found in them?

Answer 4

DNA libraries: permanent collection of genes can be obtained and maintained tehere
genomic libraries: chromosomal DNA
cDNA libraries: represent mRNA present in a given sample

Question 5

what is reverse transcriptase and complementary DNA?

Answer 5

reverse transcriptase is the enzyme that “untranscribes” RNA
cDNA is what happens after RNA has been untranscribed

Question 6

what uses can be made out of the recombinant DNA construct?

Answer 6

microarray and in situ hybridization techniques can reveal mRNA expression, co-regulation, and localization
recombinant DNA expression vectors enable regulated expression of exogenous genes and production of proteins in prokaryotic and eukaryotic cells

Question 7

what is a vector?

Answer 7

a vector can be obtained from bacteria or animal cells
designed to grow, can propagate in a cell

Question 8

what is recombinant DNA?

Answer 8

it involves fusing together two DNA fragments that are from different sources

Question 9

how does the creation of sticky ends work?

Answer 9

restriction endonucleases recognise a particular sequence in the DNA
cut the DNA in a symmetrical fashion
makes staggered cuts
the staggered ends create complementary sticky ends
vectors have only one restriction site per enzyme so that they are linearized (from a circle to a line)

Question 10

what happens after the restriction endonucleases create the sticky ends?

Answer 10

unpaired genomic fragments are ligated together
by DNA T4 ligase (comes from bacteriophage)
this process requires 2ATP

Question 11

what are the three regions of the plasmid and what do they do?

Answer 11

the replication origin
a region that carries a gene that is resistant to various antibiotic drugs
-> propagate plasmid in the presence of the drug to make sure it stays there (resistant against ampicillin)
the polylinker:
has sites for different restriction enzymes adjacent to each other
a fragment can be produced with a different restriction enzyme at each end -> unidirectionality

Question 12

how is a recombinant plasmid created?

Answer 12

the DNA is enzymatically inserted into the plasmid vector

Question 13

what is the transformation step and how is it achieved?

Answer 13

the recombinant plasmid has to go into the bacterial cell
mix E.coli with plasmids in the presence of calcium chloride (CaCl2) and/or (?) have a heat pulse
this makes the cell wall more permeable and can also create holes in the membrane
culture on nutrient agar plates containing ampicillin (the antibiotic drug)
cells that take up the plasmid survive (have the drug resistance gene) and the cells that do not take up the plasmid die

Question 14

what happens after transformation?

Answer 14

cell multiplication
the plasmids replicate inside the cells and the cells themselves replicate, each containing copies of the same recombinant plasmid

Question 15

what does reverse transcriptase do?

Answer 15

it can make a DNA copy of an RNA molecule?

Question 16

how can recombinant technology help in creating DNA libraries?

Answer 16

cut the vector with BamH1
partially digest genomic DNA with Sau3a (so that not all sites get cut)
ligate the vector with the different fragments, so that each plasmid has a different fragment

Question 17

what does in situ hybridisation reveal?

Answer 17

it reveals the spatial distribution of an RNA
the spatial distribution of the expression of a gene can be found

Question 18

what does microarray do and how does it work?

Answer 18

simultaneously measures the levels of many mRNAs?
1. the plate contains a solid support that has a known array
2. the plate either contains serum (growing) or no serum (non growing)
3. the mRNA is isolated
4. reverse transcriptase transforms it to cDNA, labelled with a fluorescent dye (red or green)
5. mix
6. hybridise to DNA microarray
cDNA will bind to the complimentary DNA
7. wash, and measure green and red fluorescence over each spot (green for the non growing, red for the growing)
8. if green= expression of the gene decreased after serum addition
if red= expression of the gene increased after serum addition
yellow/orange: roughly equal expression
blue/black: only low level expression
allows us to get a read out of the effect of serum on a large number of genes

Question 19

how can eukaryotic proteins be produced in bacteria (E.coli)?

Answer 19

with the lac promoter
on the plasmid there is the lac promoter and the lacZ gene
the lacZ gene is removed and the eukaryotic gene (as cDNA) that codes for that protein is inserted
the E.coli is transformed, and the lac promoter drives transcription
the protein can be synthesised by the bacteria
IPTG turns the promoter on or off

Question 20

what are the two ways cloned genes can be expressed in cultured animal cells?

Answer 20

transient transfection
the vector with the cDNA, the promoter and the viral origin of replication are transferred to cells by electroporation or lipid treatment
the protein is expressed from cDNA in plasmid DNA

stable transfectation (transformation)
the vector has the promoter, the cDNA and the drug resistant gene
transferred by lipid treatment or electroporation
only the cells that have the plasmid can survive
protein is expressed from cDNA integrated into the host chromosome