L7: Molecular Bio Techniques I Flashcards
What does recombinant DNA technology allow?
Allow isolation & manipulation of DNA
How is a recombinant DNA molecule created?
Isolated DNA fragments are inserted into a cloning vector
What is a cloning vector?
DNA molecule that can be introduced into a host organism & can self-replicate
What are restriction enzymes?
Function as ‘molecular scissors’ to cut DNA
How do restriction enzymes function?
Recognised specific nucleotide sequences in the DNA & cut both strands of the sugar-phosphate backbone
Define palindrome
Sequence that is read the same both ends
What do restriction enzymes leave when they are cut in a cleavage?
Sticky/blunt ends
What does the number & size of fragments of DNA depend on?
- Restriction enzyme
- Size of genome
- Abundance of each nucleotide
How are restriction maps created?
Cutting DNA with individual restriction enzymes
How is the digested DNA separated using?
Agarose gel electrophoresis which separates DNA frgaments by size
How are the DNA fragments size identified?
Comparing how far they move in the gel compared to DNA fragments of known sizes
What is bacteria/yeast used in recombinant DNA?
Replicate & amplify individual DNA fragments
What is the use of plasmids in recombinant DNA?
Used as ‘cloning vectors’ to carry DNA fragments to host
What do plasmids contain?
Antibiotic (AB) resistance genes allowing selective growth of bacteria containing plasmids
What is the role of plasmids
Carry gene for replicating their DNA
What are plasmids used for?
DNA cloning
2 different types of plasmids
1) Cloning plasmids for cloning genes
2) Expression plasmids adapted to allow gene expression
How can a DNA molecule be cloned ?
Inserting it into a plasmid cloning vector
How is a recombinant DNA molecule created?
DNA cut by same restriction enzyme will have same cohesive ends with complementry base pairs, these will anneal to one another
How is a DNA fragment cloned?
1) DNA fragment & cloning vector (e.g plasmid) cut with the same restriction enzyme
2) They have the same complementary cohesive ends
3) Cut DNA fragments & vector are mixed together
4) DNA fragments will anneal to cohesive ends in vector
5) DNA ligase ligate the DNA fragment to form recombinant DNA molecule
Where is cloned DNA amplified?
E.coli
How is cloned DNA amplified?
Individual recombinant plasmids is taken up by E.coli & using the origin of replication (ori), 100-200 copies of it is generated
What does lacZ encode?
Encodes beta-galactosidase which acts on X-gal to produce blue bacteria
Describe genomic DNA library
1) Human DNA cut with restriction nuclease to create millions of DNA fragments
2) DNA fragments inserted into plasmids using ligase to create recombinant DNA molecules
3) Plasmids is then introduced into bacteria to create a genomic library
Uses of cloned DNA
1) Identify changes in the genome
2) Characterise how genome is organised
3) Engineer organisms by transferring genes between organisms
Why can’t genomic DNA be used for studying/use in therapies?
Contains introns
Isolate mRNA where introns are removed to create cDNA
How is complementary DNA (cDNA) made?
DNA copy of a mRNA, using an enzyme reverse transcriptase
What do cDNA libraries contain?
Complementary DNA copies of the mRNA present in a cell population & represent genes being expressed in a population
Where is cDNA used?
Used in eukaryotic expression vectors as introns are removed
Does mRNA contain introns?
NO
What is a cDNA library?
Large collection of plasmids each containing a single cDNA
What do frequencies in genomic DNA & cDNA only include?
Only sequences found in mature mRNA
What are expression plasmids?
Plasmid cloning vectors that allow expression of cloned genes in bacteria
What are eukaryotic expression plasmids?
Plasmid cloning vectors that allow expresison of cloned genes in eukaryotic tissue culture cells
What is cloning vector and expression vector?
Cloning vector is a small piece of DNA maintained in bacteria
Expression plasmid used to introduce cloned DNA into bacteria to allow expression of cloned gene
Cloning vector vs Expression vector
Uses
Cloning vector used to introduced cloned DNA into bacteria to make lots of copies of insert DNA
Expression vector used to obtain gene produce of cloned DNA (protein/RNA)
-
What does nucleic acid hybridisation allow?
Allow identification of nucleic acid fragments/clones containing specific sequences
- Relies on comlpementarity of strands of nucleic acid
Explain the nucleic acid hybridisation principle
Individual strands of DNA separated by heating
- After cooling the complementary strands anneal together which ‘hybridise’ one another
Explain nucleic acid hybridisation technique
Used to identify DNA/RNA that match a specific sequence using ssDNA as a probe to identify fragments in a collection that have a sequence complementary to the probe DNA
Define probe
Single-stranded sequence of DNA/RNA used to identify specific sequences of DNA/RNA
What is the DNA probe labelled with?
Using DNA polymerase to incorportate labelled dNTPs
How can dNTPs be labelled?
Making them radioactive using 32P
OR
Attaching a fluorescent molecule
OR
Attaching digoxygenin (STEROID MOLECULE)
What is nucleic acid hybridisation used to?
To identify DNA fragments in the genome that contain specific genes
What is used in the southern blot?
DNA fragments separated by gel electrophoresis
Use of genomic southern blot
Allow identification of genomic DNA fragments generated by restriction enzymes containing a specific DNA sequence
Use of a clone blot
Allow identification of restriction fragments in cloned DNA that contain DNA sequence matching probe DNA
