L23 - Gene cloning in eukaryotes 1 Flashcards
(14 cards)
what is a YAC
Yeast Artificial Chromosome
= engineered DNA molecule designed to carry large fragments of DNA and replicate inside Yeast
describe the construction steps of a YAC
- partially digest some genomic DNA to create lots of different length fragments with sticky ends
- linearise YAC vector with the same restriction enzymes
= creates 2 arms that will ‘hold’ the insert
- insert is ligated into YAC
- YAC is transformed into yeast cells defective for tyrptophan and uracil
= meaning the cannot grow UNLESS they receive the genes from the YAC
- grow on slective media lacking tryptophan and uracil
- identify whether succesful ligation into the YAC has taken place by seeing if the ‘indicator’ gene within the MCS was disrupted
name the 2 types of vectors you use to create a eukaryotic genomic library
YAC or BAC
= plasids can only handle up to a 15 nucletide insert size
difference between genomic library an cDNA library
eukaryotic DNA contains non-coding regions
genomic libraries contain EVERYTHING - introns,exons and regulatory
= useful to study whole genome structure
cDNA libraries only contain coding regions –> reverse transcribed mRNA
= usefel to study gene expression
name the 3 types of mRNA proscessing
5’ guanine cap
3’ poly A tail
alternative splicing = different splice results produce different proteins by leaving out or keeping in different exons
describe how cDNA is made
- add TTTT primer to anneal to 3’ poly A tail
- reverse transcriptase added with dNTPs
= cDNA strand produced from mRNA template growing from TTT primer
- RNAse H added
= partially cuts and removes the hybridised mRNA = leaves over small bits
- small bits act as primers to complete the cDNA double strand with DNA polymerase
describe the simple steps to make cDNA library
- produce cDNA from mRNA with reverse transcriptase
- ligate into vectors via partial restriction digest
- transform the recombination vectors into cells
- select via selctive media (antibiotic resistance) = shows they have the vector NOT neccasarily the insert
= each colony on media will have a different length cDNA = can pick and choose for experiments
what is a ‘library’
different length DNA fragment in each colony
= can pick and choose as we like for different experiments
how can we use a cDNA library to compare different tissues or peoples gene expression
abundance of cDNA in the library for each tissue reflects the mRNA/gene expression
summarise genomic libaries in eukaryotics
genomoic libraries for eukaryotes give DNA level info –> genome and regulatory elements organisation
cDNA libraries represent gene expression/transcriptomics
cDNA produced by Reverse transcriptase
cDNA libraries from different tissues give a ‘snapshgot’ of gene expression in thar tissue at that time
= most tissue expression usaully meaured by NGS/RNA-seq
describe how you could screen a cDNA library by PCR for a sequnec of interest
- design PCR primers for cDNA of interest
- lyse colony and anneal primer for PCR
- produce amplified cDNA of interest if its present
- run on agarose gel to see if fragment of interest was present
= only tells you if a fragment of correct size is present
= use sanger or NGS to find actual sequence
how can we screen a cDNA library by hybridisation
- take nylon replica of cDNA library
- lyse cells and denature DNA to seperate strands
- add fluorescent or radioactively labelled hybbridisation probe
- hybrises to sequnce of interest if present
- radioactive signal viewed with X-ray film
= the dark spot corresponds to the replica colony taken
= go back to orignal cDNA library = the coressponding colony will have fragment fo interest
describe screening of cDNA library by expression cloning
this is about detecting a protein NOT a DNA sequence
- induce protein expression and take nylon replica
- lyse cells to release protein
- add specific antibody with flourescent tag
= flourescence indicates presence of that gene in colony
summarise screening cDNA libraies and charecterising the clones
cDNA libraries made by reverse transcriptase from mRNA and cloning into suitable vector and bacterial host cells
cDNA libraries can be screened with PCR or hybridisation of radiolabelled probes
expression libraries produce proteins that can be screened by antibodies
cDNAs can be futher analysed to see gene and protein function by PCR or cloning
= functional assay with enzymes and substrates can aslo be used to investigate as a way of screening
