Flashcards in Recombination techniques Deck (56):
How are cells grown for study?
Cells first need to be dissociated from a tissue
They are grown in vitro in culture with appropriate nutrient and factors
What are primary cultures?
cultures directly removed from tissue
What are secondary cultures?
Cultures taken from primary culture suspended in medium and allowed to propagate (passage in culture)
What is immortalized cells?
adding factors to continually make cells (making a cell line)
What can you add to cells to make them replicate over and over?
What are Embryonic stem cells?
inner cell mass of early embryo. (blastocyst)
that can be induced to differentiate into many different cell types
What can embryonic stem cells do?
they are totipotent
What is totipotent?
can differentiate into all types of cells
Have unrestricted developmental potential
What do Embryonic stem cells need to divide into different cells?
for each cell type a different stimuli is required for the cell type that is needed
How do you personalize stem cells?
1)remove nucleus from unfertilized egg
2)inject egg with nucleus from genome to be cloned (cell fusion)
3) cell division into blastocyst
4) inject blastocyst into foster mother or into culture dish(therapeutic cloning)
What can hybrid cells be used for?
Mass production of antibodies
How are hybrid cells made?
1) suspend two cell types and add a fusing agent
2) Cell fusion, a heterocaryon is made
3)selective medium allows only heterocaryons to proliferate, they become hybrid cells which then are cloned
How is an antibody made?
1) inject species with antigen
2)B-lymphocytes make antibody for X
3)mix antibody with tumor of b lymphocyte cells
4) some cells are mixed with b lymphocytes and tumor cells and produce the antibody
What is Aminopterin?
inhibitor of nucleotide biosynthesis
mutant b lymphocytes will lack the pathway for indefinite growth and use a alternate pathway
What does the B lymphocyte and tumor B lymphocytes result in?
hybridoma cells cultured in multiple wells
What is the purpose of using Aminopterin?
using it will force the b lymphocyte tumor cells to use the alternate pathway for nucleotide biosynthesis. if the alternate pathway is disabled they will not be able to grow in medium
What does b lymphocytes do after being in a culture for a few days?
they will die
How are the cells selected after producing the tumor and normal cell hybrid?
cells that are hybrids will survive only
1) tumor cells will die because they are inhibited by Aminopterin
2) regular b lymphocytes cells will die a few days in medium
3) the antibody producing hybrids can now be selected for and reproduced
Why aren't human chromosomes are studied as intact molecules?
Human chromosomes are too large to be studied as intact molecules
What is done to human chromosomes to study them?
fragmenting them at defined sequences using restriction enzymes to generate a collection of manageable pieces
What are restriction enzymes?
Endonucleases that recognize specific sequences and cut both DNA strands
What do digested DNA
Cohesive ends that can be ligated back
What are the different ends generated by restriction enzymes?
What sequence does restriction enzyme ecoR1 recognize?
5` GAATTC 3`
3` CTTAAG 5`
it cuts between the G nucleotide and the A nucleotide and leaves a 5` overhang
What is the sequence EcoRI leaves after it cleaves the DNA?
5` AATTC 3`
3`G 5` 5` overhang
What is the sequence HpaI recognize?
5` GTTAAC 3`
3` CAATTG 5`
it cuts between the A nt and T nt and leaves blunt ends
What is the sequence left by HpaI after it cleaves the DNA?
it leaves blunt ends
What is the sequence recognized by HindIII?
it cuts between the A nts and leaves a 5` overhang
What is the sequence after it is cleaved by HindIII?
it leaves a 5` overhang
What is the sequence recognized by PstI?
it leaves a 3` overhang
What is the the sequence after PstI has cleaved the DNA
it leaves a 3` overhang
What can be done with annealed cohesive ends?
they can be ligated with complementary ends and a recombinant DNA can be created
What can polyacrylamide Gel Electrophoresis (PAGE) separate?
Short fragments separated
When Nucleotides can be separated by one base pair
What can Agarose Gel electrophoresis separate?
Mid-size DNA fragments
hundreds of nucleotides
What can Pulsed -field Gel Electrophoresis separate (PFGE)?
Large size fragments
as large as 2.5 million bases
What is one way of labeling DNA fragments?
1)denature a DNA fragment
2)add short radioactive primer sequences
3) allow that DNA sequence now to be synthesized
radioactive labels will show up
What is an example of a label that is used to Tag DNA?
a specific fluorescent antibody can bind to digoxigenin and now it can be seen where the labels are bind. will be in pairs
This is known as FISH (fluorescence in situ hybridization)
How do you label the ends of DNA fragments?
radioactively label the 5` end with 32P-labeled ATO
after it has been cut by nucleases each strand will have a radioactive label
you then can see where the regulatory protein has bound
What is Southern Hybridization (Blot)?
Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
What are the steps of southern blotting?
1) run unlabeled RNA or DNA on Agarose gel. They will separate by size
2) lay a nitrocellulose or nylon membrane on top of gel. on the bottom will be a buffer. paper towels will be on top of nitrocellulose paper
3) buffer will transfer nucleotide fragments onto the nitrocellulose or nylon paper
4) remove nitrocellulose or nylon paper and place in plastic bag with radio labeled probe hybridized to separated nucleotide fragments
5) labeled probes hybridized to nucleotide fragments will show up by X ray
What is Northern blotting?
study gene expression by detection of RNA (or isolated mRNA) in a sample.
How can you separate a the mRNA in Northern blot?
by use of a poly A tail. poly T can be used to bind to it and it can be separated from all the other RNA
What do you do to the mRNA after it separated in a Northern blot?
Run it on a gel and use a probe it with what ever gene you want to look at and see how it is expressed
After the probe has tagged its target what things will it tell you?
1) In which tissues is the RNA expressed
2) What is the abundance of the RNA in the different tissues
3) is there any alternative splicing that has occurred
What is the difference of Southern and Northern blotting?
Southern uses DNA as its target and the method tells you the specific DNA sequences
Northern uses RNA as its target and it tells you the expression of RNA in tissues
What is cloning?
giving a identical copy of DNA
What is a vector?
a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA
What is done with the recombinant DNA after the foreign DNA is added to the vector?
it is put inside a bacterial cell and the bacteria cell produces hundreds of millions of copies.
the bacteria is lysed and the recombinant DNA is purified and retrieved
What type of vector is used to clone large amounts of DNA (like human DNA)?
Yeast Artificial Chromosome Vector (YAC vector)
Which three pieces is necessary for a YAC vector to replicate as an artificial mammalian chromosome?
Origin of replication
What two restriction enzymes are used to Cut a Circular YAC vector into three separate pieces?
one piece cut by BamHI is not used
one piece contains CEN, ORI, TEL
one piece just contain TEL
What is done to the human DNA to prepare it to join with the two pieces of YAC vector?
it is lightly digested with EcoR1
the complementary ends of the human DNA anneals with the complementary ends of the YAC
What is yeast ARtificial chromosome?
A yeast chromosome where a human DNA is inserted and replicated for study
How do you clone a small fragments of DNA for a human genomic library?
1) use a restriction enzyme to cut up the pieces
2) insert these fragments into separate plasmids
3) insert each plasmid into bacteria and let the bacteria multiply
How is cDNA cloning done?
2)hybridize with poly T primer
3)make cDNA copy with reverse transcriptionase
3)degrade RNA with RNase H, leave RNA primers
4)synthesize second cDNA strand from RNA primers