molecular genetics technologies Flashcards
what is type II restriction endonucleases function?
cleave DNAs at specific bases, molecular scissors
what is type II restriction methylases function?
methylate DNAs at specific bases and stops restriction enzyme cleavage
what are the three DNA polymerases’ functions?
copy DNA from 5’ to 3’ direction
eliminate error through 3’ to 5’ exonuclease activity
remove DNA ahead of them through 5’ to 3’ exonuclease activity
what is RNA polymerase function? (less imp)
RNA copy of DNA from the promoter
what is the reverse transcriptase function? (less imp)
DNA copy from RNA from 3’ end primer
what is DNA ligases function?
joins 2 DNA molecules by forming 5’ to 3’ phosphodiester bond
what is exonucleases function? (less imp)
remove nucleotide residues from DNA ends
what is the terminal transferase function? (less imp)
adds homopolmer tails to DNA
what is polynucleotide kinases function?
add phosphate to 5’ end DNA, using t4
what is alkaline phosphatases function? (less imp)
remove terminal phosphates from DNA ends
homodimer vs heterodimer
homo- protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues
hetero- protein composed of two different chains
what are the three types of ends formed by type II restriction endonucleases
(molecular scissors)
- 3’ recessed ends(longer) (5’ staggered ends(shorter))
- blunt ends
- 5’ recessed ends (3’ staggered ends)
what end, 5’ or 3’ is always phosphorylated?
5’ end
what end, 5’ or 3’ is always hydroxylated?
3’ end
what shape do the type II restriction endonuclease and DNA form
homodimer forming a 2-fold symmetric enzyme-DNA complex
what is it called when only one strand is methylated?
hemimethylated
when does intermolecular ligation occur? and intramolecular ligation? concentration wise
inter (between two separate molecules) at high DNA ligase concentration
intra (with itself) at low DNA ligase concentration– forming linear DNA
used in insertion of DNA segment in E.coli, high first and then low to finish and complete the circle
what method is used to introduce vector DNA in a plasmid in cloning?
transfection (via non viral method)
what method is used to introduce vector DNA in a phage in cloning?
transduction (via viral method)
explain the mechanism of molecular cloning
1) DNA fragment inserted into cloning site in a vector to produce a recombinant DNA molecule
2) introduced into host cell
3) replicated and amplified
4) recombinant (new) DNA passed in cell division–> colony formed
name the two pathways that λ can clone through
lysogenic pathway (λ integrates into E. coli)
lytic pathway (uses bacterias resources to duplicate and spread)
outline λ’s lytic pathway
1) theta replication when entered bacteria
2) uses ROLLING CIRCLE replication
3) gene A endonucleases cleave catenate at cos sites creating sticky ends
4) DNA packaged in new phages
what size does λ need to be do be packaged
what can be done to manipulate the size?
less than 49 Kb
large inserts cannot be packaged
non-essential regions can be removed
what is the region called that replaced the non-essential region, in lamda replacement vectors?
stuffer region, it contains restriction enzymes
what are the advantages of using a cosmic vector?
has more DNA than lamda vector ~35 Kb
no lysis occurs and colonies are produced
it exploits the cos site at high frequency
what are the two ways you can clone from a complex genome?
- constructing a genomic or cDNA library
- use PCR to amplify specific sequences of genomic DNA or cDNA
how are genomic DNA libraries constructed?
from cloning overlapping DNA fragments generated by partial digestion with restriction enzymes
then inserted in a centrifuge to separate by size and identified by hybridization/ PCR…
what are red/gam genes
is a stuffer region in lamda replacement vector
when present inhibits lytic growth
this is often replaced to allow for growth
how are genomic libraries constructed with a cosmid vector?
cell DNA prepared by partial digestion with Sau3A, fractioned into BamHI sites of cosmid vector
the colonies represent different parts of the genome
how do you calculate the number of clones needed in a certain sample to represent the genome? in a cosmid vector genomic library
Number of clones needed=ln(1-probability of getting a specific piece of DNA)/ ln(1- DNAfrag size/total genome size)
what is meant by chromosome walking
one probe detects several clones, the terminal regions of them can then detect neighboring probes and so on
in cDNA libraries how are the restriction sites added? why?
by Linkers
they improve cloning efficiency and create cohesive ends
in PCR, what are the primers called?
degenerate oligonucleotide primers
In PCR cloning, how is it directly cloned into plasmid vectors?
TOPO cloning
use vector with T overhang and topoisomerase linked with TaqPCR (has an extra A on ends)— increases efficiency
what is sanger DNA sequencing
sequencing by synthesis from DDNTPs from clones
they act as primers incorporating radioactive labels and base-specific termination
4 reactions done each with different nt
what are the principles of NGS
all direct DNA sequencing NO CLONES
DNA libraries modified by adaptor ligations and PCR
DNA fragments immobilised
no electrophoresis used
detector system involved
4 main steps in ILLUMINA NGS
1) ligate adaptors
2) cluster generation- amplifies individual DNA molecules in situ
3) sequencing and imaging
4) Data analysis
