dna techniques - clonign, gene library, plasmids Flashcards
Which of the following DNA sequences is least to be a recognition site for a restriction enzyme?
5′-GAATTC-3′ 5′-GATC-3′
3′-CTTAAG-5′ 3′-CTAG-5′
5′-GCCCCG-3′
3′-CGGGGC-5′
5′-CTGCAG-3′ 5′-CCCGGG-3′
3′-GACGTC-5′ 3′-GGGCCC-5′
OPTION C as it is not PALINDROMIC sequence
Restriction enzyme sites (specifically type II enzymes, account for almost all of those we use) are palindromic. > which means the two strands have the same sequence when read 5′ → 3′ (e.g. in the EcoRI site (answer A)) both strands read GAATTC.
The only sequence that is not palindromic is option C!
What property of eukaryotic mRNA allows it to be easily purified from sample of total RNA?
A The absence of introns.
B The lack of complex secondary structures of the types seen in tRNA and rRNA.
C The much greater variability in the size of mRNA molecules by comparison with tRNA and rRNA.
D The presence of the cap at the 5’ end.
E The presence of the poly A tail at the 3’ end.
OPTION E - only mRNA has polyAAA not the tRNA or rRNA
this helps to purify mRNA from total RNA using oligodT cellose chromatography
uses immobilised oligo-dT in a collumn to bind to the mRNA whilst the rest of RNA will elute
what is a dna clone?
DNA molecule that is genetically
identical to the DNA molecule from which it was
derived (cloning is copying)
how does genomic and cdna compare
genomic DNA can be used to create a gDNA library or to use as a template for PCR (exon+intronic DNA)
cDNA - rna that needs to be reverse transcribed. used to create the cDNA library or amplify a gene with NO INTRON (bacteria cant process introns)
how does gel electrophoresis work?
USE IT TO SEE IF THE RESTRICTION ENZYME WORKED AND QUALITY OF FRAGMENTS
separates protein/nucleic acids by size and confirmation, move from the negative to positive anode
> then we stain the gel to see the bands and use DNA ladders to estimate size of band fragment
rate migration is inversely proportional to log of size
WHAT DO TYPE 11 RESTRICTION ENZYMES RECOGNISE?
they cleave at DNA sequences which are palindromic 4-8bp long
what ends does SMA1 r.e and BAMH1 r.e create?
sma1 creates blunt ends and bamh1 creates sticky ends (where there is a 5’ or a 3’ overhang)
if my restrictiion enzyme has a frequency of cleaving 1/206 then how long would my DNA fragment be?
206bp long
pvul and xorll are isoschizomers. what does this mean?
they are restriction enzymes that cut and recognize the same sequence
a neoschizomer will recognize but cut the DNA differently
what are restriction enzymes?
site specific endonucleases which can be produced by some bacteria species
they will recognize specific DNA sites and cut them and reveals a 3’ hydroxyl group on one nucleotide and a 5’ phosphate on the other
if bacteira create their own r.e how do they avoid
Bacteria have a “restriction-modification system” with a
(ii) A sequence-specific methylase enzyme that methylates bases in the restriction enzyme target site so that it is no longer recognized.
Methylation may occur on either adenine or cytosine.
what enzyme is used to join sticky ends or blunt ends together? A RNA POL B DNA HELICASE C TELOMERASE D DNA LIGASE E PROTEIN KINASE A
OPTION D - LIGASE
can make covalent bonds between two different DNA
fragments (preferably fragments with the same sticky end).
• A 5′ phosphate group is essential for ligation to create the new phosphodiester bond. this 5’ is created when DNA is cut by r.e
why is ligation with blunt ends harder than with sticky ends
blunt ends have no intrinsic hydrogen bonding/ base pairing so efficiency of ligation with blunt ends is lower
how can we optimsise dna ligation?
DNA LIGATION NEEDED TO TRANSFORM A CELL
use sticky ends/ r.e that create sticky ends
use the optimum time and temp to ligate
> cooler temp is for blunt ends
use a ratio of 1:3 or higher of vector:target DNA fragment
WHAT IS A MIXED SITE?
its when 2 r.e recognize different sequences but create the sticky ends which are compatible with each other
but as they ligate, this new piece of DNA no longer is recognized by either of those r.e as no longer palindromic
why would we create a gDNA or a cDNA library?
if we don’t know the genomic sequence of our target gene
if we do we can go straight to PCR
what are the features of an ideal vector? (such as a plasmid)
it has an origin of replciaton
it has a unique r.e cutting site
it has a selectable marker (to select the host cells)
and it has a way to screen that the plasmid has taken up the target gen
can you explain selection
- selection - seeing if the host cell contains a plasmid
USUALLY plasmid has antibiotic resistance gene so host cells that survive will go onto the next round of screening
can you explain screening
- screening - seeing if the plasmid vector actually contains the target gene. has the host cell actually been transformed?
usually use LACZ gene which gets disrupted if there is an insert and will remain colourless if disrupted and stin blue if it is normal
name 2 methods we can transform a cell
- calcium chloride + heat shock
- electroportation which involves a brief electric
pulse (2000V)
both ways will get the recombinant DNA/ the vector into the host cell
name some other types of vectors apart from plasmids
bacteriophages
cosmids (hybrid of plasmid and phage) - 100kbp
bacteria artificial chromosome - 300kbp fragment
yeast artificial chromosome - 1Mbp fragments
how does X-gal work?
it is the substrate for LAcZ enzyme so when cleaved will turn blue
if LACZ enzyme/gene is disrupted (say there is a transgene inserted in the middle of it) then won’t be able to cleave so substrate remains colourless
what is functional compliemtnation used fot?
way to IDENTIFY a gene involved in a process e.g what is the gene responsible for arginie syntheses?
decribe how functionakl complimentation works
- create a gDNA library of all wt DNA (completely or partially overlapping sequences) using r.e
- create plasmid vectors with each of these fragment and transform mutant cells
- screen the recombinant cells and see if they can now grow on medium without arginne / has the mutation been COMPLEMENTED
- need to keep isolating these DNA fragments until a gene has been found by sequencing it! (repeat using different r.e and smaller fragments
e.coli is a common host cell used in tranformation. why do we need to use cDNA if i want to create a recobinat plant protein
as the gDNA from plants will contain introns which will not be processed by the bacteria
cDNA lacks introns so overcomes this issue
what is an expression vector
plasmid has promoter which drives expression
of the insert gene in E. coli
Promoter activity may
be constitutive
or inducible/ repressible. What does this mean?
constitutive-ly active - always on
inducible - controlled by external factros like adding chemicals