Lecture 10: Molecular Genetic Techniques Flashcards
1. DNA Cloning 2. DNA libraries 3. Nucleic Acid Detection Techniques 4. Analysis of Gene Expression
Define recombinant DNA
any DNA molecule composed of sequences derived from different sources
State the purpose of using recombinant DNA technology for DNA cloning
- Allows preparation of large numbers of identical DNA molecules-> often genes
- > allows detailed studies of the structure and function of a gene at the molecular level
Define nuclease, endonuclease and exonuclease
- Nucleases: hydrolyses ester bond within a phosphoester bond
- Endonuclease: Nuclease that cleaves phosphodiester bonds within a nucleic acid chain
- > may be specific for RNA or for ss or ds DNA
- Exonuclease: nuclease that cleaves phosphoester bonds one at a time from the end of a polynucleotide chain
- > may be specific for either the 5′ or 3′ end of DNA or RNA
Define restriction endonucleases
bacterial enzymes that recognize specific short sequences of DNA (4-8 bp) called restriction sites and cleaves both DNA strands
What type of sequences are usually in restriction sites
- Usually palindromic sequences -sequence is the same on each DNA strand when read 5’ to 3’
List the different types of restriction endonucleases
- > Type II endonucleases
- >Isoschizomers
For each enzyme, give the source microorganism, recognition site and ends product
- BamHI- Bacillus amyloliquefaciens- GGATCC- sticky
- Sau3A- Staphylococcus aureus- GATC- sticky
- EcoRI- Escherichia coli- GAATTC- sticky
- HindIII- Haemophilus influenzae- AAGCTT- sticky
- Smal- Serratia marcescens- CCCGGG- blunt
- Notl- Nocardia otitdis-caviarum- GCGGCCGC- sticky
Define cloning vector
- Cloning vector: DNA (often a plasmid) that can be used to propagate ( breed specimens) an incorporated DNA sequence in a host cell
State what vectors contain and why
- Vectors contain selectable markers + replication origins
- > allow identification and maintenance of the vector in the host
- origin of replication
Define multiple cloning sites
- Multiple cloning site (MCS or polylinker) :A synthetically generated sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules
State what type of ends can restriction endonucleases form
- Restriction endonucleases can be used to cleave DNA into defined fragments
- DNA fragments with sticky or blunt ends can be
Describe how the restriction fragment and vector join
- Covalent bonding- joins the ends of restriction fragment and vector DNA that have complimentary ends
- > restricted with the same endonuclease
Explain why 2 different restriction enzymes are used
for directionality +stop recircularisation of the plasmid
- Recircularisation- cut plasmid closes itself due to ligation
State the ratio for insert: vector molar
- 5:1 or 10:1
Define transformation
- Transformation: acquisition of new genetic material by incorporation of added exogenous, nonviral DNA (e.g. plasmid)
- Transformation- when bacteria successfully take up recombinant plasmid
Explain how bacterial transformation can happen
- In the lab this is done with CaCl2 and heat shock (42°C)
- Bacteria take up the plasmid molecule and acquire antibiotic resistance (from the plasmid)
Explain how amplification and plasmid purification occurs
- Colonies (successfully taken up recombinant plasmid) picked from the agar plate and grown in liquid broth with antibiotic
- Produces millions of bacteria all having the same plasmid
- Plasmid DNA isolated from the bacteria using methods: e.g alkalinelysis based spin-column
Explain why false positives can occur for recombinant DNA and explain how we screen for false positives
- False pos. ->because some plasmids recircularise without an inserted cloned fragment
->still have antibiotic resistance on the host - Blue/white selection- allows identification of bacteria that contain the vector plasmid WITH an insert
- Insertion of a DNA fragment-> interrupts lacZ gene
= bacteria grown in the presence of X-gal are white and not blue - White colonies selected
- Used to prepare more plasmid DNA for further analysis
Explain how vectors can be specialised for different purposes
- Shuttle vectors: can be propagated in more than one type of host cell
- Expression vectors: contain promoters that allow transcription of any cloned gene
- > designed for gene expression and protein synthesis by using cell’s machinery of desire protein
For each type of vector, give their features, isolation of DNA and DNA limit
- Plasmid- high copy number- physical - 10kb
- phage- infects bacteria- via phage packaging - 20kb
- cosmid- high copy number- via phage packaging - 48kb
- BAC- based on F plasmid- physical- 300kb
- YAC- origin+ centromere+ telomere- physical- >Mb
What are genomic libraries and why are they useful
- DNA library is a collection of DNA molecules each cloned into a vector
- The set of clones collectively represents all the DNA sequences in a genome
- Useful for representing the genomic content of simple organisms
- More useful to construct cDNA libraries for higher eukaryotes
Define reverse transcriptase
- Reverse Transcriptase: used to synthesize a strand of complimentary DNA to each mRNA molecule
Explain how cDNA clones are formed for cDNA libraries
- DNA copies of mRNAs are synthesized and cloned into plasmid vectors
- Reverse Transcriptase: used to synthesize a strand of complimentary DNA to each mRNA molecule
- RNA strand in the RNA/DNA hybrid duplex is then degraded by RNaseH or alkali
- Second-strand synthesis by DNA polymerase
- Fragments are protected by methylation at EcoRI sites
- Then synthetic linkers containing a EcoRI restriction site are added to the double-stranded cDNA molecules
- Vector + collection of cDNAs ligated
+ transformed into E. coli
= generates individual clones
Explain the variation of number of cDNA clones represented in cDNA library
- cDNA clones corresponding to abundantly expressed genes -> represented many times in a cDNA library
- cDNAs corresponding to infrequently transcribed genes ->extremely rare in cDNA library
Explain what is screening DNA libraries
- Most genomic or cDNA libraries contain 100,000s of individual clones
- Screening is required to identify clones of interest
Define hybridisation and probe
- Hybridisation: The ability of complementary single-stranded DNA (or RNA) molecules to associate specifically with each other via base pairing
- Probe: a radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment
Explain how DNA libraries are screened
- place nitrocellulose filter on master plate to pick up cells from each colony
- incubate filter in alkaline solution to lyse cells and denature released plasmid DNA
- hybridise with labelled probe
- wash away labelled DNA that doesn’t hybridise to DNA bound to filter
- perform autoradiography
- signal appears over plasmid DNA that is complementary to probe
Define in situ hybridisation and state what it is used for
- in situ hybridisation: Hybridisation of a DNA or RNA probe to intact tissue to locate its complementary strand by autoradiography (x-ray)
- Used to detect mRNA or DNA
- Hybridisation methods are used for identifying specific sequences
- in situ hybridization in whole tissues uses mRNA probes
Explain what gel electrophoresis is and give an example
- Gel electrophoresis: separates DNA fragments by size or shape/confirmation, using an electric current to cause the DNA to migrate toward a positive charge
- E,g for shape/ confirmation topoisomerases
Define and explain the process of southern blotting
Southern blotting: involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridisation with a labelled probe.
- DNA/ RNA applied to gel and electrophoresed
- buffer that had been added before electrophoresed- able to allow DNA/ RAN to blot from agrose gel to nitrocellulose filter- via capillary action
- Filter bathes in alkaline solution while blotting0- allows DNA to be denatured and stick to filter
- hybridise with labelled probe of desired sequence
- wash away unbound probe + expose to x-ray film
- develop autoradiogram
What is Sanger sequencing
- Cloned DNAs are rapidly sequenced by the dideoxy chain-termination method
Explain the difference between classic chain termination sequencing and Sanger sequencing
- Classical chain termination sequencing uses dideoxynucleotides (ddNTPs) -terminate DNA synthesis at particular nucleotides
Define primer
- Primer: A single stranded nucleic acid molecule with a 3′ –OH used to initiate DNA polymerase replication of a paired template strand
Explain the process of Sanger sequencing
- Relatively low concentration of ddATP-> incorporation of ddATP
= chain-termination- occurs at a given position in the sequence only about 1% of the time - Eventually-> reaction mixture contains a mixture of prematurely terminated daughter fragments ending at every occurrence of ddATP
- This is repeated for every ddNTP
- Radiolabelled ddNTPs-> allows detection on X-ray film by autoradiography
- Shortest travel furthest-> so read bottom up
- Fluorescently labelled ddNTPs + capillary gel electrophoresis
= allow automated, high throughput DNA sequencing
what are the limitation for Sanger sequencing
- Still difficult to parallelise
- time consuming cloning steps
- relatively large amounts of DNA are needed
Describe what the human genome project is and state its history
- The Human Genome Project -started in 1990 took:
- > 13 years
- > laboratories around the world
- > hundreds of millions of dollars
- revolution in cracking the code of life. New >£1000.00 era
Explain the overview process of next generation DNA sequencing
- DNA fragments created
- Longer fragments ligated to adapter
- PCR amplifies fragments = forms spot with many copies of same fragment
- Using PCR product as template, 1 NTP incorporated by DNAP and terminated. Then photo taken. Cycle restarted and next Np added. This is repeated
Describe the differences between Sanger and NGS DNA sequencing
- Integration: Efficiency of sequencing pipeline
- Parallelization
- Miniaturization
- Sequencing by synthesis
- reversible terminated chemistry
Explain how PCR is used in DNA sequencing
- PCR-> exponential amplification of a desired sequence
- Uses primers that anneal to the sequence of interest
- RT-PCR: uses reverse transcriptase to convert RNA to DNA for use in a PCR reaction
- Real-time/quantitative PCR: detects the products of PCR amplification during their synthesis
- > more sensitive and quantitative than conventional PCR
- PCR depends on- use of thermostable DNAP that can withstand multiple cycles of template denaturation
Define northern blotting and state what it is used for
- Northern blotting: involves the transfer of RNA from a gel to a membrane, followed by detection of specific sequences by hybridisation with a labelled probe
- Study for gene expression-> detect mRNA
Explain the process of northern blotting
- RNA applied to gel and electrophoresed
- Buffer blots RNA onto filter via capillary action
- Hybridise with labelled probe of desired sequence
- Wash away unbound probe + expose to X-ray film
- Develop autoradiogram
Explain the process of DNA microarray
- Gene expression arrays -used to detect the levels of all the expressed genes in an experimental sample
- DNA microarrays has known DNA sequences spotted or synthesized on a small chip
- Genome-wide transcription analysis is performed using labelled cDNA from experimental samples hybridised to a microarray containing sequences from all ORFs of the organism being used
- Competitive hybridisation of the red and green cDNAs to the microarray α relative abundance of each mRNA in the two samples
Explain the use of expression vectors
- Expression vectors contain promoters and ribosome binding sites (Shine-Dalgarno sequence)
= allow for efficient transcription +translation of any cloned gene - Host cell transcription and translation machinery produces protein from cloned DNA
- Most bacterial expression vectors use the lac operon promoter
- Transcription from the expression vector is induced by addition of a lactose analogue: IPTG (Isopropyl β-D-thiogalactopyranoside)
Explain what SDS Polyacrylamide Gel Electrophoresis is
- Proteins can be separated according to size by SDS-PAGE
- Gel is made from polyacrylamide- matrix through which proteins (or DNA) migrate
- Proteins are denatured prior to electrophoresis by sodium dodecyl sulphate (SDS, a detergent) and heat
= proteins are all linear -> disulfide bonds disrupted (ME, DTT) - SDS is (-) charge detergent
= gives all proteins the same charge of (-ve) - Amount of SDS molecules a protein binds α its size
- Proteins are stained with a blue dye called Coomassie blue
Explain western blotting
- Identification of the recombinant protein difficult based on size alone
- > can be detected by blotting with antibodies that is coupled to fluorophore
- transferring/blotting all of the separated proteins present in the gel onto a sheet of nitrocellulose paper
1. protein applied to SDS gel and electrophoresed
2. elctrotrasfer proteins from gel to nitrocellulose membrane
3. incubate membrane with primary antibody
4. incubate with enzyme-linked (fluorophore)secondary antibody
Explain what Epitope-Tagged Expression Vectors are describe their use
- Expression vectors can also contain an epitope tag
- Epitope tag: A short nucleotide sequence added to the start or end of the cDNA.
- Expressed protein will have a short additional peptide sequence that can encode a recognition site/ epitope for an antibody
- OR a amino acid sequence to assist purification of the recombinant protein
- Example- 6xHistidine Tag
Explain how - 6xHistidine-tagged recombinant proteins are used for
- 6xHistidine-tagged recombinant proteins purified by Immobilised Metal Affinity Chromatography (IMAC)
- Ni2+ (metal) resins are used to selectively bind (chelate) histidine
= also the tagged protein - Commercially available antibodies for recognition of 6xHistidine tags
Explain what GST is used for
- Epitope tags - allow purification of large multi-protein complexes
- Glutathione S-transferase (GST) is commonly used as an epitope tag to purify proteins
- GST binds to glutathione
- Glutathione attached to a resin
List what is Expression Vectors for Mammalian Cells
- Expression vectors contain:
- viral promoter
- viral replication origin
- polyA sequence
- ribosome binding sites (Kozak sequence)
= allow for efficient transcription and translation of any cloned gene in mammalian cells
What are expression vectors for mammalian cells used for
= allow for efficient transcription and translation of any cloned gene in mammalian cells
- Host cell transcription and translation machinery produces protein from cloned DNA
- Vectors are introduced into cultured mammalian cells by a process called transfection
Explain what is transient transfection
- Viral replication origin and promoter
- During cell division:
->plasmids not segregated properly into both daughter cells
= over time- large proportional of cells in culture won’t contain a plasmid
= hence the name transient transfection - Expression from these episomal plasmids silenced by chromatinization
Explain what is stable transfection
- Integration occurs at random sites in the genome
- Vector carries a selectable marker which provides resistance to G418
- Expression from these integrated plasmids continues for as long as the culture is maintained
Describe the various ways that CRISPR/Cas9 Targeted Genome works Editing
a. genome engineering with Cas9 nuclease
- guide-rna find specific sequence on DNA
- Cas9 cleaves dsDNA
- repair of ds break by non-homologous end joining or homology directed repair(use of donor DNA)
