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
