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BMD211-Human Molecular Biology > Lecture 10: Molecular Genetic Techniques > Flashcards

Lecture 10: Molecular Genetic Techniques Flashcards

1. DNA Cloning 2. DNA libraries 3. Nucleic Acid Detection Techniques 4. Analysis of Gene Expression

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1
Q

Define recombinant DNA

A

any DNA molecule composed of sequences derived from different sources

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2
Q

State the purpose of using recombinant DNA technology for DNA cloning

A
  • 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
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3
Q

Define nuclease, endonuclease and exonuclease

A
  • 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
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4
Q

Define restriction endonucleases

A

bacterial enzymes that recognize specific short sequences of DNA (4-8 bp) called restriction sites and cleaves both DNA strands

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5
Q

What type of sequences are usually in restriction sites

A
  • Usually palindromic sequences -sequence is the same on each DNA strand when read 5’ to 3’
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6
Q

List the different types of restriction endonucleases

A
  • > Type II endonucleases

- >Isoschizomers

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7
Q

For each enzyme, give the source microorganism, recognition site and ends product

A
  • 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
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8
Q

Define cloning vector

A
  • Cloning vector: DNA (often a plasmid) that can be used to propagate ( breed specimens) an incorporated DNA sequence in a host cell
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9
Q

State what vectors contain and why

A
  • Vectors contain selectable markers + replication origins
  • > allow identification and maintenance of the vector in the host
  • origin of replication
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10
Q

Define multiple cloning sites

A
  • 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
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11
Q

State what type of ends can restriction endonucleases form

A
  • Restriction endonucleases can be used to cleave DNA into defined fragments
  • DNA fragments with sticky or blunt ends can be
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12
Q

Describe how the restriction fragment and vector join

A
  • Covalent bonding- joins the ends of restriction fragment and vector DNA that have complimentary ends
  • > restricted with the same endonuclease
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13
Q

Explain why 2 different restriction enzymes are used

A

for directionality +stop recircularisation of the plasmid

- Recircularisation- cut plasmid closes itself due to ligation

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14
Q

State the ratio for insert: vector molar

A
  • 5:1 or 10:1
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15
Q

Define transformation

A
  • Transformation: acquisition of new genetic material by incorporation of added exogenous, nonviral DNA (e.g. plasmid)
  • Transformation- when bacteria successfully take up recombinant plasmid
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16
Q

Explain how bacterial transformation can happen

A
  • 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)

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17
Q

Explain how amplification and plasmid purification occurs

A
  • 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
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18
Q

Explain why false positives can occur for recombinant DNA and explain how we screen for false positives

A
  • 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
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19
Q

Explain how vectors can be specialised for different purposes

A
  • 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
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20
Q

For each type of vector, give their features, isolation of DNA and DNA limit

A
  • 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
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21
Q

What are genomic libraries and why are they useful

A
  • 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
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22
Q

Define reverse transcriptase

A
  • Reverse Transcriptase: used to synthesize a strand of complimentary DNA to each mRNA molecule
23
Q

Explain how cDNA clones are formed for cDNA libraries

A
  • 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
24
Q

Explain the variation of number of cDNA clones represented in cDNA library

A
  • 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
25
Q

Explain what is screening DNA libraries

A
  • Most genomic or cDNA libraries contain 100,000s of individual clones
  • Screening is required to identify clones of interest
26
Q

Define hybridisation and probe

A
  • 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
27
Q

Explain how DNA libraries are screened

A
  • 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
28
Q

Define in situ hybridisation and state what it is used for

A
  • 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
29
Q

Explain what gel electrophoresis is and give an example

A
  • 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
30
Q

Define and explain the process of southern blotting

A

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
31
Q

What is Sanger sequencing

A
  • Cloned DNAs are rapidly sequenced by the dideoxy chain-termination method
32
Q

Explain the difference between classic chain termination sequencing and Sanger sequencing

A
  • Classical chain termination sequencing uses dideoxynucleotides (ddNTPs) -terminate DNA synthesis at particular nucleotides
33
Q

Define primer

A
  • Primer: A single stranded nucleic acid molecule with a 3′ –OH used to initiate DNA polymerase replication of a paired template strand
34
Q

Explain the process of Sanger sequencing

A
  • 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
35
Q

what are the limitation for Sanger sequencing

A
  • Still difficult to parallelise
  • time consuming cloning steps
  • relatively large amounts of DNA are needed
36
Q

Describe what the human genome project is and state its history

A
  • 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
37
Q

Explain the overview process of next generation DNA sequencing

A
  • 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
38
Q

Describe the differences between Sanger and NGS DNA sequencing

A
  • Integration: Efficiency of sequencing pipeline
  • Parallelization
  • Miniaturization
  • Sequencing by synthesis
  • reversible terminated chemistry
39
Q

Explain how PCR is used in DNA sequencing

A
  • 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
40
Q

Define northern blotting and state what it is used for

A
  • 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
41
Q

Explain the process of northern blotting

A
  1. RNA applied to gel and electrophoresed
  2. Buffer blots RNA onto filter via capillary action
  3. Hybridise with labelled probe of desired sequence
  4. Wash away unbound probe + expose to X-ray film
  5. Develop autoradiogram
42
Q

Explain the process of DNA microarray

A
  • 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
43
Q

Explain the use of expression vectors

A
  • 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)
44
Q

Explain what SDS Polyacrylamide Gel Electrophoresis is

A
  • 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
45
Q

Explain western blotting

A
  • 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
46
Q

Explain what Epitope-Tagged Expression Vectors are describe their use

A
  • 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
47
Q

Explain how - 6xHistidine-tagged recombinant proteins are used for

A
  • 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
48
Q

Explain what GST is used for

A
  • 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
49
Q

List what is Expression Vectors for Mammalian Cells

A
  • 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
50
Q

What are expression vectors for mammalian cells used for

A

= 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
51
Q

Explain what is transient transfection

A
  • 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
52
Q

Explain what is stable transfection

A
  • 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
53
Q

Describe the various ways that CRISPR/Cas9 Targeted Genome works Editing

A

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)

BMD211-Human Molecular Biology (5 decks)

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