Molecular Techniques Flashcards
What is polymerase chain reaction (PCR) ?
In vitro technique to amplify a specific sequence of DNA, generating thousands to millions of copies of a particular DNA sequence within a short period of time
What are the reagents inside a PCR tube?
DNA of interest
Taq polymerase
Forward and reverse primase
dNTP
What are the three steps in PCR ? And what are their respective temperatures?
- Denaturation 95C
- Annealing 54-68C
- Elongation 72C
What happens in Denaturation step?
DNA samples are denatured at 95C to break the hydrogen bonds holding the double stranded DNA together to form single stranded DNA
Each strand will act as template for synthesis of this complementary strand
What happens in the annealing step?
Temp lowered to 54-68C for DNA primers (in excess) in PCR mixture to anneal to DNA template
Forward and reverse primers are used, binding by complementary base pairing to sequences flanking opposite ends of the target DNA sequence to be amplified
Are enzymes needed in the annealing step? Why?
Not needed
Bonds formed between primers and DNA templates are H bonds (weak)
What happens in Elongation step ?
- Temp raised to 72C - optimal working temperature of heat stable taq polymerase
- Taq polymerase attaches and will catalyse the synthesis of new complementary strand by addition of free deoxyribonucleotide (dNTP) to the 3’ end of the primer
Why are primers needed ?
To provide the free 3’ OH for the DNA polymerase to add new dNTPs to elongate the newly synthesised strand
What are the products of one cycle (3 steps) ?
Number of copies of DNA is doubles
How long is the PCR cycle repeated for?
25-30 cycles in the automated thermocycler (PCR machine) to obtain over 1 billion copies of double stranded target DNA sequence
Why is there no end replication problem in PCR ?
End replication problem : RNA primers are removed
PCR : DNA primers used to avoid the need to use DNA polymerase 1 to remove primers
Why is taq polymerase used in PCR instead of normal DNA polymerase found in our cells?
PCR steps involve high temperatures
Polymerase (protein) needs to be thermostable (since they are present in the tube from the start)
Why does taq polymerase have thermostability?
Presence of cysteine in AA sequence = more disulfide linkages formed within protein
What are the three advantages of PCR ?
- Speed and ease of use
- Sensitivity as molecular technique to clone DNA
- Robustness
How is speed and ease of use of PCR an advantage ?
- completed in relatively short amount of time
- each cycle doubles the copy number of amplified gene (2 to power of number of cycles)
- easy to setup PCR reaction and use thermocycler
Why is sensitivity as molecular technique to clone DNA an advantage of PCR?
PCR is capable of amplifying sequences from minute amounts of target DNA and can even amplify the DNA from a single cell
How is robustness an advantage of PCR?
Robustness : dependability of PCR process to continue operating well even if conditions are constantly changing
Allows amplification of DNA from various species and sources, including badly degenerated DNA or DNA embedded in medium from which conventional DNA isolation is problematic
What are the five limitations of PCR ?
- Need for prior information before PCR can be carried out
- Limitation in amount of DNA obtained
- Limitation in the size of DNA to be cloned
- Infidelity of DNA replication
- Contamination
How is need for prior info before PCR can be carried out a limitation ?
Information of base sequences flanking the gene of interest must be known before PCR to allow synthesis of specific primers at forward and reverse positions of gene sequence
Optimisation of PCR conditions : primer annealing temp, primer concentration, magnesium concentration ( needed for taq pol)
Why is limitation in amount of DNA obtained a limitation ?
Actual yield of PCR is much less than theoretical yield as amount of products at each cycle eventually levels off after many repeated cycles
Why does amount of products level off after many repeated cycles of PCR?
- some template may break down or fail to dissociate form other macromolecules during purification
- optimum temp for taq pol is 75-80C, subjecting these enz to temps higher than optimum can lead to denaturation (over time)
- as concentration of double stranded product reaches high levels, competition increases between annealing of template to primer and reannealing of the complementary template strands
- magnesium required as cofactor for thermostable DNA polymerase, determining optimum concentration used is critical to success of PCR
How is limitation in size of DNA to be cloned (amplified) a limitation ?
PCR can only be used to clone smaller DNA sequences (0.1 to 5 kb)
How is infidelity of DNA replication a limitation ?
Taq polymerase lacks a proofreading function which can lead to error during DNA replication and errors may be amplified during PCR
How is contamination a limitation ?
Due to high sensitivity of PCR process contamination from non-target DNA present in lab environment can present common problem
Non-target DNA could be amplified as well due to possibility of more than one sequence within genome being complementary to primer sequence
What is gel electrophoresis ?
An analytical technique used to separate and sometimes purify macromolecules (esp proteins and nucleic acids - DNA, RNA) that differ in size and charge
What are the principles of separation in gel electrophoresis ?
- agarose gel is immersed in electrophoresis buffer which maintains pH at constant and provides ions to carry electric current across matrix
- negatively charged DNA fragments will migrate towards positive pole (anode) when electric current applied
- agarose gel acts as molecular sieve to separate DNA fragments
- the larger DNA fragments move slower while smaller fragments move faster, molecules of the same size will migrate at same rate forming a single band
- different sized molecules will form distance bands on gel
How are DNA samples prepared for gel electrophoresis ?
DNA samples are loaded into wells of agarose gel with loading dye (combination of bromophenol blue - runs in front and xylene cyanol - run behind)
What are the two functions of loading dye ?
- Weigh down the DNA samples to ensure that it remains in the well
- To monitor the progress of the separation process so that the DNA does not overrun out of the gel - stop when bromophenol blue reaches the end of gel
Why is DNA ladder added ?
DNA ladder : mixture of DNA molecules of known sizes which can be used as molecular markers to determine size of DNA sample
DNA ladder is loaded into a separate well and run parallel and simultaneously with the other wells containing DNA samples
Why is visualisation of DNA bands needed ?
DNA bands on gel are invisible to naked eyes as DNA is colour less
How are the 3 ways DNA bands are visualised ?
- Methylene blue (non-selective)
- Ethidium bromide with UV light (non-selective)
- Nucleic acid hybridisation (selective)
How is methylene blue used to visualise DNA bands ?
Gel is stained with methylene blue which binds weakly to the phosphoric acid of DNA via ionic bonding
All DNA can be visualised under white light
How is ethidium bromide with UV light used to visualise DNA bands?
Gel is stained with ethidium bromide and visualised under UV light
Ethidium bromide
- intercalates (stack between base pairs) the DNA and makes it visible under UV
- is carcinogenic (causes cancer)
How is nucleic acid hybridisation used to visualise DNA bands ?
After carrying out southern blotting, radioactive or fluorescent labelled probes are added that will hybridise to specific DNA fragments
Only DNA containing sequence of interest can be visualised
What is hybridisation ?
DNA probes binding to target sequence to form double-stranded hybrid DNA
What are the principles of southern blotting and nucleic acid hybridisation?
- double stranded DNA samples can be made into single stranded by heating at 95C
- short, synthetic, single stranded DNA (DNA probes) with nucleotide sequences complementary to target sequence are introduced
- hybridisation
- DNA probes are labeled with radioactive elements or fluorescence markers this position of target DNA molecules are made visible
- autoradiography used to detect presence of radioactive probes
- UV light in fluorescence microscopy used to visualise fluorescent markers
What is the procedure of southern blotting and nucleic acid hybridisation?
- After running DNA fragments on agarose gel, gel is placed in buffer solution (mixture of alkali and salt) to denature DNA fragments - double stranded DNA become single stranded
- Gel is covered with nitrocellulose filter, additional absorbent papers added on top of filter - single stranded DNA is drawn up from gel to filter by capillary action
- Filter is baked at 80C so DNA is permanently bound to filter
- Filter is exposed to solution containing radioactively labelled single-stranded DNA probe - complementary bind to DNA sequence of interest
- Autoradiography : Excess DNA probe washed off and X ray film liad over filter - radioactive probe forms dark image
Sickle cell anemia chromosome
Normal : HbA HbA
Carrier : HbA HbS
Anemia : HbS HbS
What is the difference between HbA allele and HbS allele?
HbA : two DNA fragments upon digestion (1.2kb, 0.2kb)
HbS : loss of restriction site thus only one fragment upon digestion (1.4kb)
How can sickle cell anemia be detected ?
Carry out southern blotting and nucleic acid hybridisation
Anemia : 1 thick band at 1.4
Carrier : 2 bands - one at 1.4 one at 1.2
Normal : 1 thick band at 1.2
Why does digestion of DNA obtained from a cell produce different bands ?
DAN has many restriction sites and would be cut up by restriction enzyme into many different sized bands
