CLONING DNA Flashcards
How are vectors and inserts ligated to form recombinant DNA? (3 requirements)
- compatible ends formed from restriction enzyme digestion
- phosphatase vector removes 5’ end
- vector and insert should be mixed at a 1:3 ratio
3 ways of determining DNA sequencees
- Sanger sequencing
- also known as chain termination method of DNA sequencing
- based on dideoxy nucleotides that lack 3’ hydroxyl group so each base terminates at different times
= - using the mobility method based on length
- population of defined fragments of DNA labelled at one end
- sets of molecules are generated that differ in size by one base at either end
- then fractionated by agarose gel electrophoresis - next gen sequencing
- synthesis of oligos of DNA, attached to matrix
Cystic fibrosis is an autosomal ____ disease that has a mutation in the 7q31 of the ____ conductance regulator.
recessive
transmembrane
What is required for a cloning vector to be successful?
a) MCS or polylinker region that contains convenient restriction sites
b) autonomous replication
c) a selectable marker
How do we produce genomic libraries?
- cut genomic DNA with restriction enzyme
- ligate mixture of fragments into cloning vectors
- transform bacteria with a mixture of recombinants
- result will be a collection of bacteria with different plasmids containing varieties of different plasmids called A GENOMIC LIBRARY
Genomic libraries contain the whole ___.
genome
Complementary DNA is the DNA of an organism that includes only the _(a)__ DNA/ exons. To make cDNA, we need to synthesise dsDNA from __(b)__.
a) coding
b) RNA
How do we synthesise cDNA from mRNA?
- begin by binding oligo dT to mRNA molecule
- synthesise complementary strand using reverse transcriptase
- left with mRNA strand and cDNA strand
- mRNA removed using DNAseH
- chunks of RNA used to synthesise dsDNA
What is important about cDNA libraries?
they represent tissue specific expressed genes
CF phenotype affects a subset of tissues…
- lungs
- pancreas
- sweat glands
- salivary glands
Example of plasmid vectors
pUC
Why is it advantageous to use viral vectors ?
size selection, cant incorporate large amounts of DNA into bacteriophage genomes
- high frequency of infection
Why is phage lambda convenient as a vector?
- size selection (up to 50kb)
- central part of genome is not required for replication so can be completely removed and replaced with DNA of interest
- automatic packaging into phage particles for convenient storage
Cosmids are ___(a)___ between plasmids and phages. DNA can replicate like a _(b)__ and packaged like a __(c)__. _(d)__ sites signal sequences for (e) stuffing.
a) hybrids
b) plasmid
c) phage
d) cos
e) head
Cos sites stand for?
cohesive sites
How does circular DNA replicate? Forming what structure?
through rolling circle replication
- a concatemer
Chromosome walking is used to traverse __(a)_ genetic distances but is __(b)__ and labour intensive.
a) huge
b) slow
What is an expression vector?
aim to show the physical protein expression of a gene
- use prokaryotic promoters
- use variable hosts
How does chromosome walking work?
- sequential hybridisation of large fragment clones using the end of each probe
What is BAC?
bacterial artificial chromosome
What is YAC?
yeast artificial chromosome
Key points about the cDNA involved in expression vectors?
- cDNA’s are intronless
- cDNA’s use prokaryotic promoters
- cDNA’s must be insert in correct orientation
Full chromosome walking process
- RFLP probes are hybridised to a genomic library
- positive genomic fragments can be used as subsequent probes by subcloning distant end
What is a zoo blot?
southern blot from loads of different animal species
What was done to specifically find CFTR gene once cloned?
- zoo blot to see conservation of gene
- sequence and look for open reading frames
- probe northern blots and look for appropriate expression patterns eg. sweat glands
