L1-3: Molecular Cloning Flashcards
Green Fluorescent Protein (GFP)
Unique protein produced by jellyfish Aequorea victoria that emits green colour in blue/UV light
Basic components of DNA
Phosphate backbone, deoxyribose sugar, nucleotide bases
Structure of DNA
Antiparallel helix with deoxyribose sugars linked together through phosphodiester bonds.
T4 DNA Ligase
- DNA ligase frombacteriophage T4
- Catalyses the formation of a phosphodiester bond between 5’ phosphate and 3’ hydroxyl termini in duplex DNA or RNA
- It can ligate eithercohesive (sticky) or blunt endsof DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids
- Ligatesblunt-ended DNAwith much greater efficiency thanE. coliDNA ligase
DNA Gyrase
- Enzyme within the class of topoisomerase, subclass of type II topoisomerase
- Reduces topological strain in ATP-dependent manner while dsDNA is being unwound by elongating RNA polymerase or by helicase in front of the progressing replication fork
Is the origin of replication A/T or G/C rich?
A/T
DNA primase synthesises the RNA primer in a ____ direction.
5’ to 3’
Continuous DNA synthesis occurs in the ____ strand.
leading
Main differences between prokaryotes and eukaryotes
Prokaryotes: lack nucleus and organelles; single-celled; single circular chromosome
Eukaryotes: contain nucleus, and organelles; can be single-celled or multicellular; multiple linear chromosomes; much larger than prokaryotes
Cell compartments containing DNA in eukaryotes
- Nucleus
- Mitochondria
- Chloroplasts (plant cells)
T or F: only eukaryotes contain ribosomes
False.
T or F: only eukaryotes contain a cytoplasm
False.
T or F: prokaryotes do not have a plasma membrane
False.
Restriction endonucleases
Recognise and cut dsDNA at specific sequences called restriction/recognition sites
Endonucleases
Cleave within the DNA molecule at internal phosphodiester bonds
Exonucleases
Cleave at ends of DNA molecule
Recognition/restriction site
Palindromic nucleotide sequence that is recognised
Cleavage site
Phosphodiester bond that is cleaved
Frequency of restriction sites
4^n, where n = number of recognisable base pairs in the restriction site sequence
Restriction modification system
The system bacteria possess for defence against its own REs. This system is composed of a restriction endonuclease enzyme and a methylase enzyme. Each bacterial species and strain has their own combination of restriction and methylating enzymes.
Methylase/methyl transferase - an enzyme that adds a methyl group to a molecule; in restriction-modification systems of bacteria a methyl group is added to DNA at a specific site to protect the site from restriction endonuclease cleavage.
Isoschizomer
REs specific to the same recognition site e.g. Sph I, Pae I, Bbu I (5’-CGTAC/G-3’).
Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.
Neoschizomer
Recognise the same sequence but cut it differently e.g. Sma I (5’-CCC/GGG-3’) and Xma I (5’-C/CCGGG-3’)
Isocaudomer
recognise slightly difference sequence, but produces same ends e.g. Mbo I (5’-N/GATCN-3’) and BamIH I (5’-G/GATCC-3’)
Klenow fragment
DNA Polymerase I, Large (Klenow) Fragment lacks the 5’ to 3´ exonuclease activity of intact E. coli DNA polymerase I, but retains its 5´ to 3´ polymerase, 3´ to 5´ exonuclease and strand displacement activities.
DNA is ____ charged
negatively
In gel electrophoresis, DNA travels from ____ charge to _____ charge.
negative to positive
Smaller fragments of DNA used in gel electrophoresis require ______ (lower/higher) concentration of agarose.
higher
Polyacrylamide gel
Used in gel electrophoresis when DNA fragments are very small.
In gel electrophoresis, DNA fragments of 5000-12000kb require agarose concentrations of ___%.
0.6-0.8%
In gel electrophoresis, DNA fragments of 100-300kb require agarose concentrations of
1.5-2%
A restriction enzyme with a recognition site of 6bp cuts at a random frequency of ____ bp.
4,096
A restriction enzyme with a recognition site of 4bp cuts at a random frequency of ____ bp.
256
A restriction enzyme with a recognition site of 8bp cuts at a random frequency of ____ bp.
65,536
Plasmids
extra-chromosomal, autonomously replicating circular dsDNA found naturally in bacteria. Usually between 2.5-6kb in size but can be larger or smaller.
Origin of replication
Sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. For small DNAs, including bacterial plasmids and small viruses, a single origin is sufficient. Larger DNAs have many origins, and DNA replication is initiated at all of them
Replicon
Comprised of the origin of replication and all of its control elements
β-lactamases
Enzymes that provide multi-resistance to β-lactam antibiotics such as penicillins, cephalosporins etc.
Through hydrolysis, the lactamase enzyme breaks the β-lactam ring open, deactivating the molecule’s antibacterial properties.
Multiple cloning site (MCS)/Polylinker
Short DNA sequence containing two or more different sites for cleavage by restriction enzymes.
Why does self-ligation of vector plasmids occur? List methods for prevention of self-ligation and how to screen for successfully transformed colonies with successfully cloned vector plasmids.
- Use of two different restriction enzymes with different recognition sites; a plasmid cleaved with a single RE can recircularise as the two ends are complementary, producing a high number of transformants without the insert (gene of interest). Furthermore, the inserted gene of interest within the plasmid cut with two different REs has a single orientation, whereas a fragment with identical cohesive ends inserts in either orientation with equal probability.
- Partial digestion may still occur in non-ideal scenarios (vector plasmid digested by only one of the two REs). Partially digested vector plasmids will self-ligate in the presence of ligase. Hence, a selectable marker (visual selection) is used, e.g. plasmids encoding a LacZ gene surrounding the MCS/polylinker. LacZ encodes the β-Galactosidase enzyme degrades X-gal, producing a blue colour. If the gene of interest is successfully inserted within the MCS inside a LacZ gene, it will disrupt its function and blue colour won’t be produced.
- Alkaline phosphatases - hydrolase enzymes responsible for removing phosphate groups from many types of molecules. Vector plasmid treatment with alkaline phosphatases dephosphorylates the 5’ ends of vector plasmids, preventing self-ligation/recircularisation. As gene of interest contain 5’ phosphate ends, it is able to ligate into the vector plasmid.
Most restriction endonucleases recognise _______ sequences
palindromic
How are antiobiotic resistance genes used as selectable markers?
Average efficiency of bacterial transformation is extremely low (1 transformed cell per 103-105 non-transformed cells). Therefore, a selectable marker is needed to select for transformed cells (that have taken up the plasmid). Insertion of antibiotic resistance genes such as beta-lactamases that provide resistance to an antibiotic, means the antiobiotic can then be used to select for transformants that have taken up the plasmid, and have had antibiotic resistance conferred to them.
Bacterial transformation using calcium chloride
Bacterial cells are transferred to a cold (0°C) calcium chloride solution, causing the positively charged Ca2+ ions to bind to the negatively charged membrane of the bacteria, creating an electrostatically neutral environment; The negatively charged DNA is no longer repelled by the bacterial cell membrane. Low temperature congeals the membrane, stabilising the negatively charged phosphates.
Subsequently, rapidly increasing the temparature in a process called heat shocking, causing a temperature imbalance on either side of the membrane. This permeabilises the membrane, creating competent cells.
E. coli Transformation Using Electroporation
Before the electric pulse, bacterial cells are added to a solution containing the plasmids. During the electric field voltage is induced across the cell membrane, creating holes in the phospholipid bilayer. After the pulse, the phospholipid bilayer is restored with the vector plasmids inside the cell.
Key Features of Cloning Vectors (Plasmid)
- Polylinker/multiple cloning site: for introduction of DNA fragments
- Origin of replication (ori) for autonomous replication in bacterial cells.
- Selectable markers such as ampicillin-resistant genes for discrimination against empty and plasmid-containing cells
- LacZ gene or other visual markers, for visual selection between empty and recombinant vectors.
Why are long primers (50-100 nt) not used?
Long primers can form secondary structures and also annealing efficiency is reduced
Why are short primers (<10 nt) not used?
Short primers (<10 nt) are not specific enough
Optimal length of primers
18-25 nt.
Primer Design - ideal primer specifications and conditions
- Length: 18-25 bases (55-65°C)
- G/C content: 50-60%.
- G/C clamp - last 1-2 nucleotides on 3’ need to be G or C (tightly bound)
- Tm calculation: 2°C for A/T + 4°C for G/C and within 5°C of each other.
- Primer self-complementarity - hairpin loops or primer-dimers
PCR program
- Initial denaturation: 94°C - 5 mins
- Cycle 1:
- 94°C for 30s (denaturation)
- 50-65°C for 30s (melting)
- 72°C for 1min/kb (extension) - Cycle 1 repeated 25-30 times
- Last cycle:
- 94°C for 30s
- 50-65°C for 30s (melting)
- 72°C for 5min (extension)
Which end of the primer can be modified?
5’ end. The 3’ end needs to be identical to the target sequence.
T or F:
The Tm of the primer is calculated based on identical sequence plus the overhang.
False. The overhang is not included in the Tm
TA Cloning
- Subcloning technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning.
- PCR products are usually amplified using Taq DNA polymerase which preferentially adds an adenine to the 3’ end of the product. Such PCR amplified inserts are cloned into linearised vectors that have complementary 3’ thymine overhangs.
Zero Blunt PCR Cloning Kit
The Zero Blunt® PCR Cloning Kit offers an easy method for high-efficiency (>80%) cloning of blunt-end PCR products amplified with proof-reading, thermostable DNA polymerases.
The pCR™-Blunt vector contains the lethal E. coli ccdB gene fused to the C-terminus of LacZα. Ligation of a blunt PCR fragment disrupts expression of the lacZα-ccdB gene fusion, permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed when the transformation mixture is plated.
Quantitative PCR methods
- SYBR green PCR:
SYBR green binds only to double stranded DNA. Hence, the more dsDNA present in the sample (a product of PCR), more fluorescent dies will bind to DNA. The qPCR monitors DNA amplification by increase in fluorescence. - Taqman probe:
- The Taqman probe is complementary to the target DNA
- Contains a fluorescent dye on 5’ end (reporter), and a quencher on the 3’ end. When the quencher is in close proximity to the reporter, energy which would usually cause the reporter to excite and emit fluorescence is transferred to the quencher. This is referred to as fluorescence resonance energy transfer (FRET)
- As long as the probe remains intact, there is no permanent fluorescent signal (emission) from the reporter
- If the reporter and quencher are permanently separated the reporter fluoresces, producing signal that the instrument can detect. Each time a new PCR amplicon is created the reporter and quencher are split. Thus, fluorescence increases proportionally with product.
- Taq polymerase has exonuclease activity, thus has the ability to cleave the probe, separating the quencher and the reporter.
Importance for small size of vectors
- Easier to transform into bacteria
- Less chance for multiple cloning/restriction sites
- More efficient for replication than larger plasmids which take longer to replicate
Digital PCR
Digital PCR provides ultra-sensitive and absolute nucleic acid quantification:
Step 1 – The initial reaction is assembled in a single tube using the same components as qPCR
Step 2 – Assembled reaction is split into a large number of individual wells resulting in either 1 or 0 targets/well.
Step 3 – PCR amplification is performed to endpoint
Step 4 – Absolute quantification of target molecules is calculated using Poisson statistical analysis
RNA can be amplified by using _____ to generate ______ from an mRNA template
reverse transcriptase; cDNA
Southern blot hybridisation process
- DNA digestion using restriction enzymes
- Gel electrophoresis: denature with NaOH to produce ssDNA, then neutralise to pH 7.0. DNA is still single stranded.
- Blotting: nitrocellulose membrane only binds ssDNA. NC membrane will resemble pattern of gel from electrophoresis.
- Probe labelling: dsDNA probe is labeled using radioactive or fluorescent dye. dsDNA is boiled and cooled to denature and create ssDNA. Labeled probe will then bind complementary sequence on the NC membrane. Bands will appear under X-ray, mimicking location of gene of interest bands from gel electrophoresis.
The western blot uses ____ to detect _____
antibodies; proteins
South Western Blot is used for
DNA-protein interaction
North Western Blot
Proof that DNA can bind to RNA
Chain termination method
When dideoxynucleotides bind to the sequence, sequencing is arrested
Plasmid DNA normally exists in which forms
Open circular DNA, linear DNA, and supercoiled DNA - in order of appearance in gel electrophoresis
