Molecular Techniques Flashcards
What is Polymerase Chain Reaction (PCR)
In vitro technique to amplify a specific sequence of DNA, generating millions of copies of the particular DNA sequence within a short amount of time
3 step cycle (DAE) repeated 25-30 times
First step of PCR
Denaturation
- at 95C to break hydrogen bonds holding double stranded DNA together, thus forming single-stranded DNA
- each strand acts as template for synthesis of its complementary strand
Second step of PCR
Annealing
- at 54-68C
- DNA primers (in excess) in PCR mixture anneal to ssDNA template
- forward and reverse primers bind by complementary base pairing by hydrogen bonds to specific sequences flanking opposite ends of the target DNA sequence to be amplified. Anneal to 3’ end of template strands
- forward primer sequence = 5’ end (allows strand to elongate from left to right)
- reverse primer sequence = allows strand to elongate from right to left. Complementary to 3’ end
- primers provide the free 3’ OH for Taq polymerase to add new deoxyribonucleotides / deoxyribonucleoside triphosphate -> elongation
- primers prevent reannealing of 2 single stranded dna
Third step of PCR
Elongation
- at 72C : optimum temp for Taq polymerase
- Taq polymerase attaches and catalyses synthesis of new complementary strand by addition of free deoxyribonucleotides to the 3’ end of primer
- enabled by primers (provide free 3’ OH)
Why Taq polymerase instead of DNA polymerase
- Taq polymerase is thermostable, PCR involves high temp
Advantages of PCR
- Speed and ease of use
- one cycle =3-5mins, PCR is thus completed quickly (few hours)
- each cycle doubles the copy number of amplified gene = 30 cycles yields a 10^9 - fold amplification - Sensitivity as a molecular technique to clone DNA
- can work with minute amounts of DNA (eg from even a single cell) - Robustness
- dependability : keeps working even under changing conditions
- can work with DNA form various species and sources : badly degraded DNA/ DNA embedded in mediums
Limitations of PCR
- Prior info needed
- gene of interest sequence must be known prior to allow synthesis of specific oligonucleotide primers at forward and reverse positions of the gene sequence
- optimisation of PCR conditions : primer annealing temp, primer conc, magnesium conc etc - Limitation in amount of DNA obtained
- theoretical yield > actual yield (plateauing effect)
- some of template may break down/ fail to dissociate from other macromolecules during purification
- enzyme denaturation (Taq polymerase exposed to high temp above optimum 75-80C)
- reannealing of template: as concentration of double stranded product reaches high levels, competition increases between annealing of template to primers and reannealing of complementary template strands
- magnesium conc must be optimal (Taq polymerase is a magnesium-dependent enzyme) - Limitation in size of DNA to b cloned
- only can close smaller dna sequences in the 0.1-5kb range (unlike cell-based cloning using plasmid vectors) - Infidelity of dna rep
- Taq polymerase lacks proofreading function - Contamination
- PCR is highly sensitive= may amplify non-target DNA
Applications of PCR
- Determination of viral load/concentration and viral genotype
- Identifying disease organisms that are difficult to culture (viruses)
- DNA fingerprinting for paternity testing
- Forensic science with small samples/genetic fingerprinting
- Cloning genes
- Can be coupled with Reverse Transcription Polymerase Chain Reaction (RT-PCR) to study gene expression
What is Gel Electrophoresis (definition + principles)
Analytical technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids that differ in size and charge
Gel is immersed within electrophoresis buffer : maintains pH at relatively constant value + provides ions to carry electric current across matrix
Samples : DNA is negatively charged , migrates to positive anode
Agarose gel : molecular sieve (larger DNA molecules travel slower) = diff sized molecules form distinct bands in gel
Diff sized molecules form distinct bands in gel, rate of movement (dist travelled in given time) is inversely proportional to size of dna fragment
Gel electrophoresis (preparation and visualisation)
- Addition of loading dye
- DNA samples are loaded into well of a gel along with loading dye
- loading dye : weighs down DNA sample so it remains in well
: monitors progress of separation process so DNA does not overrun out of gel (DNA in gel is invisible) - Addition of DNA ladder for determination of size of DNA molecules in the bands
- DNA ladder : mixture of DNA molecules of known sizes
- DNA ladder is loaded into separate well, run parallel and simultaneously with the other wells
- visual comparison - Visualisation of DNA bands through addition of stains or probes
- Methylene Blue : binds weakly to phosphoric acid of DNA via ionic bonding. All DNA bands can be visualised under white light
- Ethidium Bromide w UV light : intercalates the DNA and makes all bands visible under UV light. Is a carcinogen
- Nucleic acid Hybridisation : only DNA band(s) containing sequence of interest can be visualised
Why does digestion (DNA cut into many fragments by restriction enzymes) of DNA obtained from a cell produce different bands (DNA Fragments of diff sizes)
DNA has many restriction sites (specific sequences) distributed randomly along dna = cut up by restriction enzymes into many different sized bands
Practical steps in carrying out gel electrophoresis (8 steps)
- Obtain DNA samples (any cells that contain nuclei)
- Addition of loading dye to dna samples
- Using a micropipette, DNA molecules are loaded into wells at one end of agarose gel, which is placed inside an electrophoresis chamber filled with an electrophoresis buffer
- DNA ladder is loaded into one of the wells, run parallel and simultaneously to other wells
- Negative Electrode placed on side of electrophoresis chamber closer to samples, positive electrode on other end = electric field set up across gel
- Negatively charged DNA fragments move towards anode, speed depends on size
- Electric supply is switched off when loading dye has travelled about 2/3 of gel
- Gel is stained for visualisation of DNA bands
Southern blotting and Nucleic Acid Hybridisation (definition + principles)
- Allow for visualisation of specific DNA fragments after separation by gel electrophoresis
- Southern Blotting : transfer of denatured DNA fragments from agarose gel after gel electrophoresis to a nitrocellulose membrane. Followed by use of radioactive/fluorescent DNA probes to hybridise to and detect specific nucleic acid sequences
- Double stranded DNA -> single stranded (at 95C/high pH>13)
- DNA probes labelled with radioactive elements/fluorescent markers: short, synthetic, single stranded DNA w nucleotides complementary to target sequence bind to target sequence to form a double stranded hybrid DNA (Hybridisation)
- Autoradiography : detect presence of radioactive DNA probes
- UV light in fluorescence microscopy : detect presence of DNA probes with fluorescent markers
Procedure of southern blotting and nucleic acid Hybridisation (7 steps)
- Agarose gel containing digested DNA is placed in mixture of alkali and salt to denature DNA fragments : double stranded -> single stranded (ssDNA)
- Gel is covered with nitrocellulose filter. Additional absorbent papers placed on top of filter. ssDNA is drawn up and transferred onto nitrocellulose filter through capillary action
- Filter is baked at 80C (permanently bind dna to filter)
- Filter is exposed to solution containing radioactively labeled ssDNA probe, probe binds by complementary base pairing to DNA sequence of interest (Hybridisation)
- Excess dna probe is washed off. Photographic / X ray film is laid over filter (autoradiography)
- Size of DNA bound by probe is determined using DNA ladder
- Once desired DNA is located, repeat electrophoresis and remove desired DNA (corresponding position of dna band on photographic film with that on the gel)
How gel electrophoresis, southern blotting, & nucleic acid hybridisation can be used to detect inheritance of Sickle Cell Anaemia
- use restriction enzyme Mstll on chromosomes containing HbA allele (normal) and HbS allele (SCA)
- HbA = 1.2kb + 0.2kb dna fragment
- HbS = 1.4kb dna fragment only de to loss of one restriction site
- restriction fragment length polymorphism (RFLP) : variation in sizes of restriction fragments when dna from diff ppl is subjected to same restriction enzyme
- 2 alleles can be easily distinguished if differences in be sequence results in loss/gain of restriction sites = diff restriction/dna fragment lengths
PROCEDURE
1. Separate dna fragments using gel electrophoresis
- 2 dna samples (normal and SCA) treated separately w Mstll restriction enzyme
- Detection of dna fragments using SB & NAH
- SB: after GE, dna from gel is denatured and transferred to nitrocellulose filter
- filter is immersed in solution containing radioactive probes, viewed over an x-ray film in autoradiography
- specific radioactive labeled probes are synthesised that Hybridisierung specifically to a sequence near the haemoglobin allele
