dna techniques - clonign, gene library, plasmids

Question 1

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′

Answer 1

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!

Question 2

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.

Answer 2

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

Question 3

what is a dna clone?

Answer 3

DNA molecule that is genetically
identical to the DNA molecule from which it was
derived (cloning is copying)

Question 4

how does genomic and cdna compare

Answer 4

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)

Question 5

how does gel electrophoresis work?

Answer 5

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

Question 6

WHAT DO TYPE 11 RESTRICTION ENZYMES RECOGNISE?

Answer 6

they cleave at DNA sequences which are palindromic 4-8bp long

Question 7

what ends does SMA1 r.e and BAMH1 r.e create?

Answer 7

sma1 creates blunt ends and bamh1 creates sticky ends (where there is a 5’ or a 3’ overhang)

Question 8

if my restrictiion enzyme has a frequency of cleaving 1/206 then how long would my DNA fragment be?

Answer 8

206bp long

Question 9

pvul and xorll are isoschizomers. what does this mean?

Answer 9

they are restriction enzymes that cut and recognize the same sequence

a neoschizomer will recognize but cut the DNA differently

Question 10

what are restriction enzymes?

Answer 10

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

Question 11

if bacteira create their own r.e how do they avoid

Answer 11

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.

Question 12

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

Answer 12

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

Question 13

why is ligation with blunt ends harder than with sticky ends

Answer 13

blunt ends have no intrinsic hydrogen bonding/ base pairing so efficiency of ligation with blunt ends is lower

Question 14

how can we optimsise dna ligation?

Answer 14

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

Question 15

WHAT IS A MIXED SITE?

Answer 15

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

Question 16

why would we create a gDNA or a cDNA library?

Answer 16

if we don’t know the genomic sequence of our target gene

if we do we can go straight to PCR

Question 17

what are the features of an ideal vector? (such as a plasmid)

Answer 17

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

Question 18

can you explain selection

Answer 18

1. 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

Question 19

can you explain screening

Answer 19

2. 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

Question 20

name 2 methods we can transform a cell

Answer 20

1. calcium chloride + heat shock
2. electroportation which involves a brief electric
   pulse (2000V)

both ways will get the recombinant DNA/ the vector into the host cell

Question 21

name some other types of vectors apart from plasmids

Answer 21

bacteriophages
cosmids (hybrid of plasmid and phage) - 100kbp
bacteria artificial chromosome - 300kbp fragment
yeast artificial chromosome - 1Mbp fragments

Question 22

how does X-gal work?

Answer 22

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

Question 23

what is functional compliemtnation used fot?

Answer 23

way to IDENTIFY a gene involved in a process e.g what is the gene responsible for arginie syntheses?

Question 24

decribe how functionakl complimentation works

Answer 24

1. create a gDNA library of all wt DNA (completely or partially overlapping sequences) using r.e
2. create plasmid vectors with each of these fragment and transform mutant cells
3. screen the recombinant cells and see if they can now grow on medium without arginne / has the mutation been COMPLEMENTED
4. need to keep isolating these DNA fragments until a gene has been found by sequencing it! (repeat using different r.e and smaller fragments

Question 25

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

Answer 25

as the gDNA from plants will contain introns which will not be processed by the bacteria

cDNA lacks introns so overcomes this issue

Question 26

what is an expression vector

Answer 26

plasmid has promoter which drives expression

of the insert gene in E. coli

Question 27

Promoter activity may
be constitutive
or inducible/ repressible. What does this mean?

Answer 27

constitutive-ly active - always on

inducible - controlled by external factros like adding chemicals