1
Q
what are the the 5 in vitro techniques for manipulating DNA
A
○ PCR
○ Electrophoresis
○ Hybridisation
○ Molecular cloning
○ Targeted mutagenesis
2
Q
what is biobrick assembly
A
- genetic engineering and assembling of biobricks
- inserted into vector or chromo
- transform into model microbe
- products
3
Q
what are the “biobricks”
A
promoters
enhancers
sensors
RBs
terminators etc
4
Q
what is a thermocycler
A
automated PCR maching
5
Q
what does the thermocycler require
A
Requires template DNA, DNA
polymerases (Taq and Pfu) , DNTPs, buffer
6
Q
why are Taq and Pfu used in PCR
A
theyre the most thermostable DNA polymerases
7
Q
what is PCR used for
A
○ Medicine - diagnosis
○ Consumer genomics
○ Food and agriculture
○ Forensic sciences
8
Q
what are the steps to PCR
A
- Denatured by heat - 95 deg
- Primers and flanking sequences
- Annealing (primers bind DNA)
- Add thermostable DNA polymerase - extends primers
- Heat and cool and repeat many times
9
Q
Which DNA polymerase does proofreading in PCR
A
Pfu
10
Q
what are PCR variations (2) and how do they work
A
- Reverse transcriptase PCR (RT-PCR)
○ Detects gene expression and intron free eukaryotic genes
○ Converts RNA to complimentary DNA (cDNA) - Multiplex PCR
○ Multiple targets at once.
11
Q
what are the steps to RT-PCR
A
§ Add oligoDT primers
§ Reverse transcription to sscDNA
§ Degrade RNAs
§ Contine with typical PCR and amplify gene of interest
12
Q
what is synthetic DNA
A
- Short DNA molecs made chemically
- Multiple oligonucleotides can be added together
13
Q
how does DNA serparate in agrose gel
A
- Seperates based on size and chage
- Nucleic acids migrate towards +ve electrode
- Small molecs move faster and further
14
Q
agrose gel is stained with _______ which fluoresces DNA with _____, and have restriction enzymes that _________________________________
A
ethidium bromide
UV light
cut DNA at specific sites
15
Q
what is nucleic acid hybridization
A
Lab method used to detec nucleic acid sequence within complex mixture
16
Q
what is nucleic acid probes
A
short, labelled, single stranded DNA
17
Q
what are the steps to nucleic acid hybridization
A
- isolate DNA
- Denature dbl stranded DNA and combine with probes
- If DNA probe finds gene of interest it will bind and light up
18
Q
how do we interpret southern blots
A
- Lit vs unlit blots
- Probe can be RNA or DNA
- Thick/Dark bands=probe binding (Target sequence present)
19
Q
whats southern blot vs northern blot
A
southern: DNA is in the gel and probe is either
northern: RNA is in the Gel. Probe is either
20
Q
how is hybridization used today
A
- gene expression and profiling
○ RNA –> cDNA –> binds complimentary DNA –> reveals genes that are on - Diagnostics
○ Probes detect specific pathogens - Environmental & Forensic Work
21
Q
what is FISH
A
- Fluorescent in situ hybridization (FISH)
○ Fluorescent probes bind target sequence in cell
○ Detects specific species or gene in samples
○ Fluorescents light up in different colours
22
Q
what is PAGE
A
Protein Analysis - Polyacrylamide Gel Electrophoresis (PAGE)
- Proteins separated by size (SDS-PAGE) or charge (native PAGE)
23
Q
TF we can do PAGE without a stain
A
F
24
Q
what is a western blot
A
done after page
○ Protein transfer to solid matrix so u can stain and visualize
○ Antibodies detect protein antigen
○ Confirms where protein is and the size of it
25
what are the specific purposes of PCR, gel electrophoresis, hybridization, FISH, PAGE
PCR - Amplify DNA
electrophoresis - Separate fragments
Hybridization - Detect specific sequence
FISH - Locate gene in cell
PAGE - Separate proteins
26
what is gene cloning
Movement of gene from original source to small manipulatable genetic element (VECTOR)
27
what is a vector
Vector is anything that will accept foreign gene for cloning
28
what are the 4 enzymes for cloning and what do they do
○ Restriction endonucleases
§ Cuts DNA at specific sites
○ DNA ligase
§ Joins 2 strands together
○ Reverse transcriptase
§ DNA from RNA
○ DNA polymerase
§ Extend or repair DNA
29
restriction endonucleases are ____ in eukaryotes, common in _______, protect proks from ___________, and are the key to ___________ and _______
rare
bacteria
hostile foreign DNA
manipulating DNA and cloning
30
how do cells protect their own DNA
Methylates their own DNA to prevent being chopped up
31
in PCR, primers bind to specific target sequences on separate DNA strands. DNA polymerase extends from these primers along the template strand. So why don't we get amplification of the entire DNA strand in the first cycle?
-The long, first-cycle products do not get amplified exponentially because they lack a primer-defined end.
DNA polymerase DOES extend along the entire template strand from where the
primer binds. 1st cycle: extension creates long, variable-length products
32
what happens in the first 3 PCR cycles
Cycle 1: Creates long products of indefinite length
Cycle 2: Long products from Cycle 1 become templates. The opposite primer now
binds and creates products bounded on both ends by primer sequences
Cycle 3+: These defined-length fragments amplify exponentially, while long products
increase only linearly
33
what is a type 2 restriction endonuclease
is an enzyme used by bacteria that cuts DNA at a specific recognition sequence.
○ Cutting results in 2 sticky end or maybe 2 blunt ends
- Recognizes palindromes
34
what is a palindrome
DNA or RNA sequence that is the same being read from 5-->3 or 3-->5
35
what is EcoR1
a restriction endonuclease enzyme isolated from strains of E. coli
- Cuts and makes 4 nucleotide sticky ends with
complementary 5’ end overhangs of AATT.
- Ligation by DNA ligase can rejoin the
two sugar-phosphate backbones
36
why do we need DNA methylation
○ After modification DNA cant be cut
37
why do we modify DNA
○ Control gene expression
○ Protect host DNA
38
TF Each restriction enzyme is partnered with
a corresponding modification enzyme
that shares the SAME recognition
sequence
T
39
what are the steps to gene cloning (overview)
1. Isolation and fragmentation of source DNA
2. Insertion DNA fragment into cloning vector
3. Introduction of cloned DNA into host organism – usually by transformation
4. Expression of cloned gene and selection and
verification of recombinants
40
why do plasmids make good vectors
- Small and easy to isolate
- Replicate independantly
- High copy #
- Selectable markers
- Vectors transfers to host
41
whats Puc19
a plasmid cloning vector
42
Puc19 contains an _____________ gene and ______ reporter, also contains ____________ (MCS) inside lacZ gene
ampicillin resistance
lacZ
multiple cloning site
43
how do we create recombinant DNA
Cut vector and foreign DNA with same restriction enzyme
2. Mix –sticky ends anneal
3. Seal with DNA ligase
4. Transform recombinant DNA into host organism
44
what is blue-white screening
Plate on medium with antibiotic
and X-gal
45
When β-galactosidase cleaves_____, it releases an indole derivative that oxidizes to form a ______________, used to identify colonies where ____________ (blue) vs. ______________ (white).
X-gal
blue insoluble pigment
lacZ is functional
disrupted by DNA insertion
46
TF in terms of blue white screening,
Blue = recombinant clone → lacZ disrupted by inserted DNA.
White = vector without insert → lacZ intact.
F
Blue = vector without insert → lacZ intact.
White = recombinant clone → lacZ disrupted by inserted DNA.
47
LacZ encodes ________, ____+____ produces
blue colour if functional
β-galactosidase
Β-gal + X-gal produces blue colour if functional
48
in confirming and analyzing clones what are the checking for correct Inserts
methods
- Amplify insert
- Restriction digest analysis
- DNA sequencing (most common)
- Hybridization with labelled probe
49
what are the Protein Expression Testing methods
If foreign gene is expressed, detect by antibody (Western blot) or enzyme activity
50
what are the ideal host characteristics and an example
○ Grows rapidly
○ Not pathogenic
○ Genetically stable
○ Replicates vector
- Escherichia coli
51
what are expression vectors for
- Designed to optimize expression of foreign genes
- Expression vectors must ensure mRNA is efficiently translated; needs appropriate RBS and start codon
- Remove eukaryotic introns (via cDNA)`
52
what are T7 expression vectors
a system used in bacteria to make large amounts of a specific protein.
53
what is a DNA library
a collection of cloned DNA fragments stored in bacteria or vectors so scientists can study genes
54
what are the 2 types of DNA libraries
Genomic Library
cDNA Libra
55
what does the genomic library contain
All genes (introns + regulatory regions)
- presents the entire genome
56
what does the cDNA library contain
No introns, only exons
- Only genes that are actively expressed in the cell
57
what are the uses of genomic library and the cDNA library
genomic: Study genome structure
cDNA: Study gene expression
58
What enzyme converts mRNA → cDNA?
Answer: reverse transcriptase
59
What starting molecule is used to make a cDNA library?
mRNA.
60
what is site directed mutagenesis vs Cassette Mutagenesis
- Site-directed mutagenesis is a method used to change a specific nucleotide in a gene using PCR
- Cassette mutagenesis replaces a short piece of DNA with a synthetic DNA fragment (cassette).
61
what are the key takeaways to these concepts
- Restriction enzymes -____________
- Ligase & vectors - ____________
- Screening - ____________
- Expression systems - ______________
- Mutagenesis - __________
Precise DNA cutting
Joining and carrying DNA
Finding successful clones
Producing proteins
Studying function
Microbiology (BMSC 210)
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