Molecular genetics tools Flashcards
(22 cards)
3 tools8
Enzymatic tools
Molecular tools
Biological tools
Enzymatic tools
Restriction endonucleases
- Enzymes that cut double stranded DNA at specific sequences
- at a position either within or outside the recognition site.
- release cohesive/ sticky ends and blunt ends
Enzymatic tools
Restriction endonucleases
Sticky ends
Eco RI
cuts leaving exposed bases
Enzymatic tools
Restriction endonucleases
Blunt ends
Smal
no exposed bases
Enzymatic tools
5 modifying enzymes
- Methyltransferases
- Nucleases: (DNases, RNases)
- DNA ligases
- Polymerases: (DNA and RNA polymerases)
- Reverse transcriptases
Enzymatic tools
modifying enzymes
Methyltransferases:
4 points
- Catalyse the transfer of a methyl (CH3) group to DNA bases.
N6-Methyladenine
N4-Methylcytosine
C5-Methylcytosineu - Used to block restriction sites.
- Approximately 1% of DNA bases undergo DNA methylation
- Involved in different functions: epigenetics.
Enzymatic tools
modifying enzymes
Nucleases:
3 points
- Enzymes that cleave randomly nucleic acids.
- Deoxyribonucleases, DNases: nucleases that cleave single stranded or double stranded DNA.
Endonucleases: cut inside the sequence.
Exonucleases: cut from the extremities - Ribonucleases, RNases: nucleases that cut RNA
Enzymatic tools
modifying enzymes
DNA ligases:
3 points
- DNA ligases catalyze the formation of a phosphodiester bond between the 5’ phosphate of one DNA fragment and the 3’ hydroxyl of another.
- 2 types:
ATP-dependent DNA ligases
NAD-dependent DNA ligases - T4 DNA ligase is used in cloning to ligate DNA fragments.
Enzymatic tools - modifying enzymes
DNA Polymerases :
3 points
- They copy a DNA strand into another DNA strand
- C-ter [domain]. large fragment: Klenow fragment two functions:
DNA polymerisation: base extension in the 5’ to 3’ direction.
Sequence editing and proofreading repairing mistakes by 3’ to 5’ exonuclease activity.
- N-ter [domain]. small fragment has: 5’ to 3’ exonuclease activity.
Enzymatic tools - modifying enzymes
Three main types of DNA polymerases in bacteria:
5 points
- DNA pol. I: main enzyme for DNA replication in bacteria. The DNA polymerases used in PCR belong to this group
- DNA pol. II: involved in DNA repair
- DNA pol. III: involved in DNA replication
- Processivity: number of nucleotides added to the new
strand per second. - Fidelity: rate of errors (wrong nucleotides added).
Enzymatic tools - modifying enzymes
RNA polymerases
4 points
- They transcribe single stranded DNA into RNA
- Prokaryotes:
The same RNA polymerase produces messenger RNA and non coding RNA (rRNA, tRNA, sRNA). - Eukaryotes:
RNA polymerase I: Large ribosomal RNAs.
RNA polymerase II: Messenger RNA.
RNA polymerase III: transfer RNA and small RNA.
Mitochondrial and chloroplastic RNA polymerases.
- In the lab T7 RNA polymerase is used to produce RNA from cDNA.
Enzymatic tools - modifying enzymes
Reverse Transcriptases
4 points
- RNA-dependent DNA polymerase
- Transcribe single-stranded RNA into single-stranded complementary DNA (cDNA).
- Used mainly by retroviruses:
HIV, Human Immunodeficiency Virus.
M-MLV, Moloney Murine Leukemia Virus.
AMV, Avian Myeloblastosis Virus - In the lab Reverse Transcriptases are used to produce in vitro cDNA from RNA
3 Molecular tools
- Vectors
- Probes
- Oligonucleotides
Molecular tools
Vectors
3 points
- They are small DNA molecules having regulatory and coding sequences.
- Foreign DNA can be inserted into them.
- They are used as carriers of foreign DNA into host cells.
Molecular tools
Vector characteristics:
4 points
- Origin of replication: replication of the vector, together with the foreign DNA fragment inserted into it.
- Genetic markers: selection of cells which have taken up the plasmid DNA.
- Multiple cloning site: a site where DNA is inserted
- Transfer DNA (some vectors): transfer a gene into a target genome. Vector DNA can be used as a DNA vaccine.
Molecular tools
Vectors - Plasmids:
3 points
- Double stranded circular bacterial DNA used for molecular cloning to
amplify or express insert DNA into bacterial hosts. - Phagemids or phasemids:
DNA cloning vectors derived from phage DNA and
containing an origin of replication. They are used
to amplify insert DNA via bacteriophage
replication into host cells. - Cosmids
Minimal phage vectors lacking the origin of replication
Molecular tools
Vectors - Bacterial Artificial Chromosome: BAC
3 points
1.Large DNA vectors engineered from the F plasmid which behaves like a chromosome.
- They are used to carry large insert DNA up to 300 Kb and
are used to create and store genomic libraries. - They are very useful in
genome studies.
Molecular tools
Vectors - Yeast Artificial Chromosome: YAC
2 points
- YAC vectors act like real chromosomes in yeast
and can store very long DNA fragments (over 150 kb in size). - Used in genomic studies
Molecular tools
Molecular probes
2 points
- Labelled polynucleotide DNA or RNA fragments, variable in size (100-1000 bp), natural or synthetic.
- Used for detection of DNA or RNA targets present in complex samples via hybridization by sequence complementarity.
Molecular tools
Oligonucleotides (oligos)
3 points
- Short (6-60 nucleotides) oligonucleotide sequences.
- Single strand oligos: used as primers for DNA and RNA amplification
- Double strand oligos: used as adapters that are ligated to DNA fragments to facilitate cloning and other applications.
6 Biological tools
- Bacterial cells
- Yeast cells
- Insect cells
- Plant cells
- Bacteriophages
- Viruses
3 Uses of biological tools
- Used as hosts to amplify DNA
- Used as hosts to store DNA.
- Used as vectors to transfer DNA to host organisms.
