Week 1 basic techniques in molecular biology Flashcards
How are double stranded dna molecules held together?
by hydrogen bonding
what do each of the single strands have?
directionality
the two dna strands are…
…antiparallel
What can dsDNA molecules be separated into?
two strands
How are dsDNA molecules separated into two strands?
heating
How are the two DNA strands reannealed?
by cooling
annealing is…
…base specific
what is meant by annealing is base specific?
strands find their complementary sequence on reannealing
How are dsDNA molecules cut?
with restriction endonucleases
What do restriction endonucleases recognise?
specific sequences (to cut the dna)
many bacterial cells have …
…plasmids
what can plasmids of bacterial cells provide?
genetic material to confer resistance / benefit to the cell.
what is dna a polymer of ?
nucleotides.
what can plasmids be used in?
molecular genetics and cloning
What are restriction endonucleases used to do?
used to insert DNA into plasmids
what does dna, a polymer of nucleotides, consist of?
adenine, thymine, cytosine, and guanidine.
what bonds are weaker, hydrogen or covalent?
the hydrogen bonds are weaker
where are hydrogen bonds in dna?
between the base pairs holding the two nucleotide chains / strands of dna together
what kind of backbone does dna have?
sugar - phosphate backbone
DNA molecules are ….
… double stranded
the two strands of dna pair together by…
…complementary sequences forming the pairs
why is dna referred to as a polymer?
meaning it consists of repeated subunits – molecules – in this case
nucleotides.
the DNA polymer chain is formed
by …
…the sugar part of one nucleotide being covalently bonded to the phosphate group of the next nucleotide (phosphodiester bond) – and so on.
in double stranded DNA A always pairs with…
…T
C always pairs with…
….G
the two strands are paired because of…
…hydrogen bonds (H bonds)
How many H bonds between A and T base pairs and C and G base pairs?
there are 2 H bonds between each A and T base and 3 H bonds between each C and G base
covalent bonds (electrons shared) are […..] bonds
covalent bonds (electrons shared) are strong bonds
hydrogen bonds (attracted) are…
…weak bonds
the dna strands can be easily separated because…
…the bonds are weak
double strand melted apart into single strands which can then be used as a…
…template
why is the fact that dna strands can be separated important?
- important in gene expression (its how genes are transcribed)
- in dna replication
- crucial in molecular biology.
the double helix can be ‘melted’ forming…
…two single stranded dna polymers
single stranded DNA molecules
will pair up again because…
…this is energetically favourable
pairing is…
…base specific
single stranded DNA molecules with
complementary sequence will …
…come back together to form double stranded DNA molecules
base pairing is dynamic. What does this mean?
when you put single stranded DNA together the molecules are constantly moving and interacting with each other
where single dna strands interact with non-complementary base sequences the stability of the double helix is …
…weak and little energy is required to separate the strands
where single strands of DNA interact with complementary sequence the double helix is …
… stable and a lot more energy is required to break the strand
Restriction endonucleases represent a kind of…
… bacterial immune system
Bacteria are constantly under attack from viruses called…
…bacteriophage
What does a phage do?
The phage insert their own DNA into a
bacterial cell in order to hijack the cell’s
metabolism and replication machinery to copy the virus
Bacteria have enzymes that…
…recognise small DNA sequences in the virus DNA and cut them
Bacteria have enzymes that recognise small DNA sequences in the virus DNA and cut them.
How do bacteria protect their own dna?
they protect their own DNA by having
it methylated at this sequence
what happens to a DNA molecule when cut with the restriction endonuclease BamH1?
The DNA at the BamH1 site is ‘cut’ – importantly it cuts between the two G bases on each strand.
What is left after a DNA molecule is cut with the restriction endonuclease BamH1?
the cut leaves overhanging ends of single-stranded DNA – these
are called ‘sticky ends’
what do the sticky ends of cut single strands of dna?
will base pair together again
Restriction endonuclease recognition sites tend to be…
…palindromic
Restriction endonuclease recognition sites tend to be palindromic. What does this mean?
they read the same backwards and forwards
Note that in this case we say they are palindromic as the top strand reads the
same as the bottom strand when read in the same direction that the DNA
strand goes.
a palindromic sequence in DNA is one
in which the…
…5’ to 3’ base pair sequence is identical on both strands.
different enzymes are used for…
…different things.
Restriction endonucleases allow us to…
… cut DNA between base pairs.
where do names of restriction endonucleases come from?
from the name of the bacteria it was found in.
To clone and express a protein from a gene of interest we need to…
…isolate the gene and pop it into an vector ‘plasmid’ so that it can be expressed.
The plasmid contains an….
… inducible promoter region to allow for expression of the gene.
When the plasmid is transfected
(transferred) into E. coli cells we can …
…induce the cells to make the protein
by ‘turning on’ this promoter.
What are the restriction endonucleases used to do?
1) Cut the DNA of interest to insert
2) To cut the plasmid vector.
The ring is the…
…plasmid
the red portion is…
…the dna sequence/gene of interest, whcih is inserted into the vector plasmid
bacterial genomes are…
…small and circular
how many chromosomes bacteria got?
a single chromosome
the genome size of bacteria is…
… much smaller than that of eukaryotes
bacteria has no…
…nuclear membrane
bacteria have no nuclear membrane. The chromosome is organised as a…
…nucleoid, but is free in the cytoplasm.
all essential genes of bacteria are contained within the…
…chromosome
in bacteria, there is little…
…repetitive DNA
what is the typical chromosome size of bacteria?
a few million base pairs and consists of a few thousand genes mostly involved in cellular metabolism
many bacterial cells also have…
…plasmids
what is a plasmid?
plasmids are extrachromosomal DNA containing non-essential genes
How do plasmids replicate?
plasmids may replicate
independently or may integrate
into the bacterial chromosome and
be inherited as episomes
for this module, the plasmids we are interested in replicate as…
…small circular DNA molecules
gene function of resistance plasmid?
resistance to antibacterial agents.
examples of resistance plasmid?
-Rbk of Escherichia coli
-other bacteria
gene function of fertility plasmid?
conjugation and dna transfer between bacteria
example of fertility plasmid?
F of E.coli
gene function of killer plasmid?
synthesis of toxins that kill other bacteria
example of killer plasmid?
Col of E.coli for colicin production
Gene function of degradative enzymes?
enzymes for metabolism of unusual molecules.
example of degradative plasmid?
TOL of Pseudomonas putida for toluene metabolism.
gene function of virulence plasmid?
Pathogenicity
Example of virulence plasmid?
Ti of Agrobacterium tumefaciens, conferring the ability to cause crown gall disease in dicotyledonous plants.
plasmids are involved in carrying…
…specific traits
plasmids are involved in carrying specific traits – usually these traits are…
…not essential in perfect growing conditions but are essential in certain environments (eg - presence of an antibiotic).
what are the different types of plasmid?
- Resistance
- Fertility
- Killer
- Degradative
- Virulence
the presence of an antibiotic resistance
plasmid in a bacterial cell exposed to that antibiotic will mean the difference
between…
…life and death
the presence of an antibiotic resistance
plasmid in a bacterial cell exposed to that antibiotic will mean the difference
between life and death – hence there is a …
… large selection pressure meaning those cells with the plasmid are strongly favoured. The bacteria with the plasmids are stronger.
What can plasmids best be viewed as?
“selfish DNA” entities.
Why are plasmids viewed as selfish dna?
they replicate themselves – they confer an advantage where they are present and because of that advantage they are able to replicate more – you can compare this to the way a virus replicates – it doesn’t ‘care’ about its host but seeks to just replicate
What group of antibiotics does penicillin belong to?
β-lactams
What are β-lactams secreted by?
secreted by fungi of the Penicillium genus.
what is the name of the resistance gene of β-lactams?
bla (beta lactamase)
How do β-lactams work?
by inhibiting cell wall synthesis
β-lactams work by inhibiting cell wall synthesis – specifically they…
…block the action of PBP (penicillin binding protein).
What is penicillin binding protein responsible for?
this is the enzyme responsible for the peptide crosslinks between adjacent chains of peptidoglycan – penicillin blocks PBP from synthesising crosslinks – because the wall isn’t constructed properly the cell lyses.
What is the name of a typical E.coli plasmid?
pBR322
What is any cell containing pBR322 resistant to?
both ampicillin (a kind of pencillin) and tetracycline
any cell containing pBR322 is resistant to both
ampicillin (a kind of penicillin) and tetracycline.
What is this resistance due to?
the resistance is due to 2 genes - bla/ampR (ampicillin) and tetR (tetracycline).
What is the ‘ori’ part of the plasmid?
where the DNA replication begins, allowing the plasmid to replicate itself.
this is a high copy number plasmid – typically there may be…
…several hundred pBR322 plasmids in each bacterial cell.
during cell division as the cell divides almost invariable there will be a number of
plasmids in each daughter cell just by…
…chance
plasmids have been engineered to be useful in…
…gene cloning.
What is pUC19 ?
a standard gene cloning vector.
What does pUC19 have?
- a gene for ampicillin resistance (ampR or β
lactamase - bla). - an mcs
- a lacZ gene
What is an mcs?
a multiple cloning site (mcs)
What is a multiple cloning site (mcs) full of?
short sequences that are recognised by
restriction endonucleases.
What does a lacZ gene produce?
a blue colouration when the bacteria are grown in the presence of a sugar called X-Gal.
The purpose of the ori sequence is that the plasmid needs to…
…replicate in the cell (when bacteria divide the daughter cells will each inherit a number of plasmid molecules).
What does the multiple cloning site (mcs) contain?
several sites recognised by different
restriction endonucleases – the sequence of that of pUC19 is above.
the sites in the mcs are…
…unique within the plasmid - nowhere else in the plasmid sequence do these sites occur.
remember that the plasmid is a…
…circular DNA molecule.
Remember that the plasmid is a circular DNA molecule – so if the plasmid molecule is cut with BamH1 then …
… it is cut at one place only
what happens to the plasmid molecule when
cut with the restriction endonuclease BamH1?
as a result of the digest, overhanging ends of single-stranded DNA are produced –
these are the ‘sticky ends’.
single-stranded DNA pairs with…
…other single stranded DNA molecules with complementary sequence.
as a result of the digest overhanging ends of single-stranded DNA are produced – these are the ‘sticky ends’
now remember that single-stranded DNA pairs with other single stranded DNA molecules with complementary sequence.
because of this we have a means of…
…inserting a new piece of DNA into the cut
BamH1 site.
now if a gene that we are interested in has a BamH1 site either side of it then…
…this can also be cut.
So if we mix together our cut plasmid DNA and our cut gene of interest what will happen?
by the process of complementary base pairing we can get the gene we are interested in joining onto the plasmid DNA.
by complementary base pairing of the cut plasmid DNA and the gene DNA we only have…
… weak hydrogen bonds formed.
by complementary base pairing of the cut plasmid DNA and the gene DNA we only have
weak hydrogen bonds formed
in order to create a stable new recombinant plasmid we need to make sure that…
… the single strands are COVALENTLY joined.
by complementary base pairing of the cut plasmid DNA and the gene DNA we only have
weak hydrogen bonds formed
in order to create a stable new recombinant plasmid we need to make sure that the single
strands are covalently joined
this is done by…
… ligation
What is the enzyme DNA ligase?
the enzyme DNA ligase is a naturally occurring
enzyme involved in joining together pieces of
DNA – by adding this it ‘fixes’ the join so that
covalent bonds form over the chimeric site
What does ligation require?
ligation requires ATP - (it is energy expensive).
How is ATP used to drive the DNA ligase reaction?
1) Ligation requires ATP
2) ATP broken down to AMP which associates with DNA ligase
3) Recognises breaks in the DNA strand.
4) Catalyses the formation of a phosphodiester bond
5) Ligase and AMP then dissociate
What do DNA ligase catalyse?
the formation of phosphodiester bonds.
how can we use plasmids in molecular biology?
refer to powerpoint slide 35
Whats MLF1?
A protein in E.coli - (Myeloid Leukaemia Factor 1).
MLF1 (Myeloid Leukaemia Factor 1) is thought to be an…
…oncoprotein thought to be involved in the determination of blood cell phenotype and is associated with blood cancer.
GST is referred to as a…
…GST fusion tag
A gene already expressed by the
plasmid pGEX4T-1 is called…
GST (Glutathione S transferase)
GST (Glutathione S transferase) is an…
… enzyme used in cell protection.
When inserted into the plasmid our
gene of interest becomes fused to the …
… known GST gene.
When both the MLF1 and GST protein are expressed we can isolate our protein of
interest using…
…this GST tag / protein
But there are many steps we have to take before we can isolate a protein.
The first step is to…
…prepare and get MLF1 into the vector plasmid (pGEX4T-1).
Where does the MLF1 DNA come from in the first place?
Extract RNA from human cell lines (generate cDNA amplify through PCR).
How do we isolate lots of DNA?
Insert into a plasmid
How do we know that DNA is correct?
DNA sequencing.
