Genetic Engineering Flashcards
Name some types of proteins
Structural proteins, enzymes, signalling proteins
What is the function of southern blotting
Detect/visualise DNA in homogenate
Describe homogenate solutions
cellular/organ structure destroyed
Describe in situ structure
cellular/organ structure intact
How is DNA detected/visualised in situ
chromosomal painting/spreads
How is RNA detected in homogenate
Northern blotting
What does western blotting detect
proteins in homogenate
What does imunno-histochemistry detect
proteins in situ
How is RNA detected in situ
in situ hybridisation
What is a disadvantage of using homogenate
requires larger quantity of tissue
What is a disadvantage of in situ methods
require tissue processing
What is useful about in situ techniques
determine sub-cellular location
What are the techniques of blotting
repeated freeze/thaw, mechanical devices, detergents
What is the first step of the blotting technique
DNA is in a gel and separated by size
What is the second step of the blotting technique
transfer out of gel - blotted by electric field or capillary action
What is the third step of the blotting technique
detection with labelled probe
what are the factors affecting DNA migration
DNA size, DNA shape, Gel type, Gel concentration
What type of gel is best for larger fragments of DNA
Agarose
What are the main two types of gel
agarose or polyacrylamide
What does a western blot rely on
principle of specific antigen-antibody interaction
What does the western blot identify
presence of protein, size and relative abundance
What does stability of hybridisation of DNA or RNA rely on?
degree of match between target and probe
What does in situ hybridisation of RNA rely on
principle of hybridisation of complementary probe to target RNA
What can in situ detection of RNA locate
RNA sequences within a tissue
What can in situ detection of RNA detect
mRNA expression
Describe correlation between signal intensity and expression in in situ detection of RNA
As signal intensity increases, level of expression increases
What are the purposes of DNA sequencing
predict function of DNA sequence, identify differences, confirm sequence of engineered DNA
How are nucleotides linked
phosphodiester bond between 5’prime phosphate to 3’prime hydroxyl
What are nucleotide terminators tagged with
fluorescent label
What size fragments run fastest
small
What are the advantages of DNA sequencing
deep sequencing, whole genome in one day, RNA sequencing
What are the purposes of restriction enzymes in DNA sequencing
enable analysis of DNA, enable DNA engineering
What does ligase catalyse
formation of phosphodiester bond
What are the characteristics of restriction enzymes that are useful for DNA engineering
cut at specific sequence, produce compatible cohesive ends
What does PCR require
small amount of DNA, known starting sequence of DNA, DNA polymerase, dNTPs
Where are DNA primers derived from in PCR
start and end
Which DNA end is sense
5’ prime end
Which DNA end is anti-sense?
3’ prime end
What is the first step of PCR
denaturation of template DNA - 95C
What is the second step of PCR
annealing of primers to template DNA - 55C
What is the third step of PCR
Extension - elongation of primers till end of template - 72C
What makes it possible for PCR to modify DNA
Primers are incorporated
What are primers used for in PCR
add suitable restriction enzyme sites - enable DNA engineering
what is a vector
DNA molecule that is maintained & replicated naturally by host organism
What does DNA cloning involve
inserting piece of DNA into a host vector
What are examples of host organisms used for DNA cloning
bacteria & viruses
What are plasmids
circular DNA - naturally found vectors in bacteria
What are the properties of plasmids
maintain and duplicate themselves
What do plasmids contain
origin of replication, anti-biotic resistance gene, restriction enzymes
What does the restriction enzyme site facilitate in plasmids
insertion of DNA insert
What are the two types of plasmid?
Cloning plasmid, and expression plasmid
What is the difference between cloning and expression plasmid
expression - more complex, has a promoter
Where does the expression plasmid insert DNA
downstream of the promoter
What are the two types of DNA library
genomic, cDNA libraries
What do genomic libraries contain
DNA sequence of organism
What are cDNA libraries derived from?
mRNA - different stages of organism
What do cDNA libraries represent
expressed/transcribed part of the genome
TRUE or FALSE - you can only have one cDNA library per organism
FALSE
What are the two types of target identification
microarrays, yeast-2-hybrid screen
What do RNA microarrays compare
transcribed genes between two tissues E.G. healthy & damaged
What do RNA microarrays rely upon
principle of hybridisation between mRNAs to probes
How many probes fo RNA microarrays need
enough to represent ALL known genes
What do RNA microarrays detect
mRNA
Where is the yeast-2-hybrid screen performed
yeast
What do you need for a yeast-2-hybrid screen
protein of interest
What does the yeast-2-hybrid screen rely on
principle that genes are transcribed from promoters
What do you fish for in a yeast-2-hybrid screen
proteins that interact with protein of interest
What is expression of genes dependant on
close proximity of transcription factor to promoter region (activation domain)
What does fusion between POI and binding domain require
expression plasmid, useful yeast promoter
What can genetic screening be used for?
diagnostic and predictive for mutations/disease
What can point mutations lead to
abolition of restriction sites
Why are changes in restriction sites a marker for mutation
they are very specific
What are mutations in restriction sites called
restriction fragment length polymorphism
Name two other techniques of target validation
gene knockdown using siRNA, gene knockout in transgenic mice
what are siRNAs
small interfering RNAs
What are siRNAs used for
reduce expression of specific genes
Describe the term knockdown
partial loss of function
What pathway does gene knockdown use
endogenous RNA interference pathway
What do cells use the endogenous RNA interference pathway for
regulate own gene expression
What is DICER?
an enzyme that cleaves free microRNA
What does DICER produce?
small double stranded fragments
What is RISC
RNA-induced silencing complex
What does RISC use to get to target
one strand of microRNA as a guide
What does RISC degrade
target mRNA
What are the two methods of gene knockout in transgenic mice
pronuclear injection, gene targeting
Where is foreign DNA injected during pro-nuclear injection
pro-nucleus of fertilised ova
Which method of gene knockout creates chimeric mice
Gene targeting
Where is the transgene introduced in gene targeting
totipotent stem cells
What are VNTRs
variable number of tandem repeats
How many VNTR variants do we have
2
Which technique of gene targeting is heritable
Gene knockout
