Recombinant DNA and molecular techniques part 1 Flashcards
List the 4 major steps involved in the Isolation and analysis of nucleic acids
- Extraction of DNA
- Nucleic acid analysis using UV spectrophotometry
- Gel electrophoresis of DNA
- Visualizing DNA molecules in an agarose gel
List the 7 major steps to extract DNA from cells and tissues
- Grind tissue with liquid nitrogen in the presence of lysis buffer (e.g. buffer containing SDS).
- Treat tissue or cell lysate with protease.
- Extract protein with phenol / chloroform.
- Repeat extraction with chloroform.
- Precipitate DNA with 100% alcohol.
- Spin down DNA pellet.
- Wash with 70% alcohol
UV spectrophotometry theory to detect nucleic acids
Nucleic acids absorb most strong at 260nm
Absorb UV light most strongly at wavelength 260 nm due to interaction between UV light and the ring systems of purines and pyrimidines
What wavelength do protein absorb UV best?
Proteins absorb most strongly at 280nm
Quantifying nucleic acids formula
OD260 X 50 (mg/ml) X Dilution factor = ? mg/mL of DNA
What does OD260 of 1.0 corresponds to
- 50 ug/ml for pure double stranded DNA
- 37 ug/ml for pure single stranded DNA
- 40 ug/ml for pure RNA
How to calculate sample purity for DNA
Pure DNA has an OD260/280 of 1.8
Lower ratios below 1.8 indicate protein contamination
Pure RNA has a ratio of 2.0
DNA denaturation (melting)
- Heat treatment causes weak hydrogen bonds of double helix to break, the helix unwinds and strands separate.
GC clamp
- GC bonds are more stable because GC base-pairs have three hydrogen bonds and AT base-pairs have only two hydrogen bonds.
• DNA with greater proportion of GC pairs than AT pairs require higher temperature in order to be denatured completely.
Melting temperature Tm
Mid-point of the curve i.e. point at which 50% of DNA strands are unwound or denatured. Curve OD260 at y axis and temperature at X axis.
As temperature increases, OD260 also increases
The higher tm have the same OD 260 as the lower tm at a higher temperature.
DNA renaturation
Reverse of denaturation process
(Single-stranded DNA re-associate to form double-stranded DNA)
If DNA that has been denatured thermally is cooled slowly. random collisions between complementary strands will result in their re-association.
Agarose gel electrophoresis
Electrophoresis is the separation of fragments by causing them to migrate through pores, under influence of an electric field.
Why DNA moves through an electric field?
DNA is negatively charged due to tis phosphate group.
When a current is applied, DNA moves from the cathode to the anode (positively-charged pole)
List the 3 factors affecting DNA mobility through agarose gels
Agarose concentration
Molecular size of DNA
DNA conformation
Agarose concentration
Lower the agarose concentration of the gel, the larger the DNA sizes that can be analyzed
Molecular size of DNA
Smaller molecules migrate at a faster rate through the gel than larger molecules
DNA conformation
Supercoiled DNA moves faster than relaxed DNA because it is tightly coiled and hence more compact
Supercoiled DNA vs linear DNA
Supercoiled DNA migrate at a faster rate through the gel than linear DNA
DNA ladders
- Contain DNA fragments of known molecular weight
- Used as reference to estimate molecular weight of
DNA fragments of interest
Visualizing DNA molecules in an agarose gel using ethidium bromide staining
- Intercalating agent -> EtBr binds DNA in between the
stacking of base-pairs - The stained DNA bands are visualized by means of
fluorescence under UV light - Mutagen
Plasmids vs Chromosomal DNA
Plasmids have no machinery to replicate, transcribe and translate on their own
Restriction Enzymes
Restriction endonucleases
What are restriction enzymes
Enzymes, found in bacteria and archaea, that recognize specific short nucleotide sequences within double stranded DNA
What do restriction enzymes do
Can cleave DNA backbone by making two incisions, once through each sugar-phosphate backbone of the DNA double helix. Note they cut phosphodiester bonds.
Restriction process
Restriction enzymes selectively cut up foreign
DNA in a bacterial host.
It is a defense mechanism against invading foreign DNA such as viral DNA by cutting it up.
How is the host DNA protected by restriction enzyme?
Host DNA is methylated by a methylase to protect it from the restriction enzyme’s activity.
how do restriction enzymes know where to cut
They bind to and cut at specific recognition sites called palindromic sequences.
What is a palindromic sequence
A palindromic sequence is a sequence of dsDNA that is the same whether it is read from top or the bottom strand (in the 5’ to 3’ direction)
If your flip the strand direction, it will still be read in the same.
What are the 2 types of DNA ends created by restriction enzymes
Sticky ends and blunt ends
Blunt ends
Blunt ends are non-cohesive ends, since there is no unpaired DNA strand fleeting at the end of DNA, made by a straight cut into the DNA
Sticky ends
The sticky ends have unpaired DNA nucleotides on either 5’- or 3’- strand, known as overhangs
What can sticky ends do?
The sticky ends that can anneal with each other,
even if the two fragments are from different sources.
Restriction mapping
The process of identifying the position of restriction sites on a fragment of DNA.
Restriction mapping use for large DNA fragments
Restriction mapping are used to provide additional markers that can be used in linkage studies to locate the map position of a gene.
Restriction mapping use for other small cloned fragments of DNA, plasmids or PCR products.
Once restriction sites are identified and mapped, the presence or absence of these sites can be used for a variety of things including the identification of biological strains, markers of genetic diseases and presence or absence of cloned inserts in a vector.
How to construct a restriction map?
Two portions of the DNA sample are individually digested with different restriction enzymes.
A third portion of the DNA sample is double-digested with both restriction enzymes at the same time.
Each digested sample is separated using gel electrophoresis with a DNA ladder, and the sizes of DNA fragments are recorded.
The length of each DNA fragments depend on the position of restriction sites, each restriction site can be mapped according to the fragment length.
PCR
PCR can amplify a DNA sequence to produce multiple copies of the DNA
List the 5 components of PCR
- Thermostable DNA polymerase called Taq polymerase
- Primers
- PCR buffer and magnesium salt
- Template (target DNA sample)
- each of the 4 nucleotides (dNTPs)
List 3 major steps in PCR
- Denaturation
- Primer annealing
- Extension
Denaturation in PCR
Hydrogen bonds that hold the double stranded DNA template together are broken.
Primer annealing in PCR
Primer bind to a specific location on the single stranded DNA template
Extension in PCR
Thermostable DNA polymerase adds nucleotides
complementary to those on the DNA template at the 3’ OH end of the primer
Cycle 1 in PCR
Step 1 Denature DNA
Step 2 Anneal protein
Step 3 Extend primers
Products of 1st cycle is 2 new DNA molecules
Cycle 2 in PCR
Step 1 and 2 Denature and Anneal new primers
Step 3 Extend primers
Product of 2nd cycle is 4 new DNA molecules
Typical timing and temperatures in a PCR reaction
94oC for 3 min (initial denaturation step)
94oC for 1 min (denaturation step)
58oC for 1 min (annealing step)
72oC for 1 min (extension step)
72oC for 5 min (final extension step) and store at 4oC
Steps 2 to 4 is repeated 30 or more cycles
PCR end product
The gene of interest is exponentially amplified after >30 cycles.
2 to the power of 35
2 to the power of n cycles
PCR for diagnostic purposes
- Many diseases are accurately diagnosed with PCR e.g. HIV, tuberculosis, SARS, COVID-19
- Uses specially designed primers for each disease and carry out the PCR reaction
- After the PCR reaction, run an agarose gel to check for desired PCR band (DNA fragment)
- Desired PCR band indicates presence of the disease
Cloning vectors
- > A plasmid that is engineered (some useful features) to carry foreign DNA into a host cell (bacteria or yeast).
- > It replicates inside the cell, using the machinery of the host cell.
List 2 important components cloning vectors
- > contain a genetic marker for easy detection of recombinant plasmid
- > contain a multiple cloning site, where a large number of unique restriction sites has been engineered to allow easy cloning of genes.
List 5 examples of cloning vectors
Plasmid - 0.1 -10 kb Lambda phage - 8 - 20kb Cosmid - 35 - 50kb Bacterial Artificial chromosome 75 - 300kb Yeast Artificial chromosome 100-1000kb
PCR summary
The mixture of taq polymerase, deoxyribonucleotides, DNA primers.
Heating to break hydrogen bonds in DNA to form single stranded molecules. It is cooled to allow primers to anneal to each end of the segments to be copied
Taq polymerase synthesize the complimentary strand of DNA using primers as starting point
The temperature is raised again to separate the DNA strands and then lowered sufficiently to allow primers to attach.
Taq polymerase then synthesize another set of new complimentary strand.
The multiple cycles of amplification can be achieved using a thermal cycler, a instrument that automatically increase and reduce temperature at appropriate time intervals.
DNA primers
Short pieces of single stranded DNA, designed to flank the target DNA region.
What are recombinant DNA techniques
- Comprises of a vast and diverse combination of techniques to construct recombinant molecules which contain DNA sequences derived from more than one source.
- Or simply the artificial joining of DNA from different
biological sources.
DNA primers function
- The primer pairs are used to hybridize with the sample DNA and define the region of the DNA that will be amplified.
- The starting point of DNA synthesis
DNA primer chemical function
PCR primers is to provide a “free” 3’-OH group to which the Taq polymerase can add dNTPs.
Lis the 5 components for cloning a gene
- DNA fragment to be cloned - cut with restriction enzymes to have ends compatible with the vector
- Vector cut with the same restriction enzymes
- DNA ligase and ligation buffer to join DNA fragment and vector together
- Host cell - usually E.coli, where recombinant vectors are introduced to the cells to obtain large quantities of recombinant DNA
- Selection medium - depends on the antibiotic resistance gene and selection marker on the vector.
List the 7 steps in cloning a gene using plasmid, blue-white screening
Cut the plasmid unique restriction site and chromosomal DNA with the same restriction enzymes.
Mix the plasmid and Chromosomal DNA together to allow sticky ends to base pair.
Add DNA ligase to covalently link a piece of chromosomal DNA into plasmid
Mix the recombinant plasmid with E.coli cells that have been treated with agents that make them permeable to DNA
Plate the recombinant cells on a selective media, which can contain IPTG, X-gal, ampicillin and incubate overnight.
The white colony in blue-white screening contains a recombinant vector with an insert and blue colony in blue-white screening contains re-circularized vector without an insert
Each bacterial colony is derived from a single cell, hence all cells in a colony are genetically identical.
Note for recombinant plasmid situation, 3 things can happen
- Gene of interest is inserted into the plasmid
- Other DNA fragments are inserted into the plasmid
- Plasmid re-circularize without an insert.
Blue-white screening
- > Vector carrying a lacZ gene with an incorporated Multiple Cloning Site (MCS) can be used for blue white screening.
- > The lacZ gene code for b-galactosidase, which breaks down lactose.
- > X-gal, an analog of lactose is added to the agar plate.
- > In the absence of a DNA insert, b-galactosidase is produced by the lacZ gene and it breaks down X-gal to give a blue color. (failed recombinant plasmid)
Heat shocking in plasmid gene cloning
Facilitate uptake of recombinant vector into the cell.
The cells are then cultured to allow resident DNA molecules to replicate and form copies.
List the 3 steps in cloning of PCR products in TA cloning
- PCR products amplified using Taq polymerase contain
an A base at the ends - The PCR cloning vector pGEM-T contains terminal T at the cloning site which can base-pair with the PCR
product - DNA Ligase covalently joins the PCR product to the
pGEM-T
Taq polymerase and pGEM-T
Taq polymerase forms an A base at the ends, pGEM-T have T terminal at its cloning site.
