Molecular genetic techniques Flashcards
The melting temperature of DNA mainly depends on… ?
GC content (b/c form 3 H-bonds instead of 2 btw AT)
What are phosphodiesterases?
also called nucleases
→cleave phosphodiester bonds btw nucleotides
- DNases if specific for DNA
- RNases if specific for RNA
What is the difference btw endo- and exonucleases?
- endonucleases: able to cleave phosphodiester bonds within the nucleic acid
- exonucleases: only able to cleave terminal phosphodiester bonds of nucleic acid, either cut in 5’→3’ or 3’→5’
What are site-specific DNA methylases?
Where can they be found?
only in bacteria
→ methylate bacterial DNA to distinguish it from foreign DNA
What are bacteriophages?
How do they incorporate their DNA into their hosts?
viruses specific to bacteria
- open bacterial genome
- insert their genome, ends resealed
- either via homologous base pairing, or
- synthesize site-specific proteins that connect to bacterial DNA if no base pairs matching
- bact. DNA linearized out
What are restriction enzymes?
also called restriction endonucleases
excise non-methylated DNA in bacteria as defense mechanisms against bacteriophages, i.e. restrict their growth
DNA sequence:
- blunt ends
- overlapping sticky ends
Explain the mechanism of restriction endonucleases.
cleave both DNA strands at specific 4-8 bp short palindromic sequences, so called restriction sites
→ create either blunt or sticky ends
- blunt ends: smooth ends
- sticky ends: ends are overlapping
How does recombinant DNA technology exactly work?
- restriction enzymes used to excise specific nucleotide sequence from any DNA strand
- inserted into vector
- vector introduced into bacteria DNA
→ DNA naturally amplified
Explain the mechanism of complementary sticky end ligation.
complementary sticky end ligation
native sticky ends are used to insert restriction fragment into vector
- bacterial restriction endonuclease creates complementary sequence on the restriction fragment and the vector
- complementary base pairing btw restriction fragment and vector
- ligated by T4 DNA ligase (hydrolysis of ATP → AMP)
What is homopolymer tailing?
used to ligate blunt ended restriction fragments to vectors (instead of sticky ends)
terminal transferase adds nucleotides to vector DNA and restriction fragment, creating sticky ends
→ then ligated by T4 DNA ligase
What are chimeric molecules?
molecules (i.e. nucleic acids) that contain sequences from two different species
What is a cloning vector?
What are general requirements of cloning vectors?
DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell
- needs ori so it can be replicated in bacterium
- needs selectable markers, so cells containing the vector can be identified
- needs spec. cleavage site for each RE used
NOTE: different vectors used in prokaryotes and eukaryotes
What is the most common type of bacterial vectors?
Characteristics.
plasmids
- small, circular dsDNA molecules carrying genes for antibiotic resistance
- have own ori, replicate independently from bacterial genome
List other bacterial vectors. What are their advantages?
- phages (bacterial viruses): have linear DNA
- cosmids: plasmids containing some viral λ-DNA
- .. artificial chromosomes: bacterial BAC, yeast YAC
→ can carry incr. amount of nucleotides
(YAC can carry largest DNA fragments)
What are viral vectors?
vectors used in eukaryotes
- adenoviral vectors
- retroviral vectors
- lentiviral vectors (group of retroviral vectors)
- adeno-associated viruses (AAV)
NOTE: limited in size of DNA sequences that can be inserted
What is an episome?
genome outside the bacterial genome
→ replicates independetly (e.g. plasmids)
What are genomic libraries?
How are they created?
total genomic DNA from a single organism is stored in vectors
DNA cleaved by REs → inserted into plasmids → plasmids introduced into bacteria
What are cDNA libraries?
How are they created?
DNA complementary to fully transcribed mRNAs (after splicing, does not contain introns), stored in vectors
mRNA is extracted → reverse transcriptase used to produce DNA copies (cDNA) of mRNA → inserted into plasmids → plasmids introduced into bacteria
What is an expression vector?
vector designed for gene expression in cells
→ contains entire gene (cDNA) that codes a specific protein
often contain highly inducible promoters