Methods in Molecular Biology and Genetic Engineering Part 1 Flashcards
utilizes laboratory-based technologies to manipulate DNA
genetic engineering
used as a tool to analyze gene structure and expression, changes in the DNA content of bacteria and eukaryotic cells by directly introducing cloned DNA to be apart of the genome
Recombinant DNA
what are the 2 essentials of a molecular biologist’s toolkit?
restriction endonucleases and cloning vectors
allow DNA to be cut into precise pieces
restriction endonucleases
such as plasmids or phages used to carry inserted foreign DNA fragments
cloning vectors
-enzymes that degrade nucleic acids, opposite of polymerase
-hydrolyze or break an ester bond in a phosphodiester linkage between adjacent nucleotides in a polynucleotide chain
nucleases
can hydrolyze internal bonds within a polynucleotide chain
endonuclease
acts at the end of a chain and hydrolyze from that end position
exonuclease
recognition site and cleavage site are the same
type II restriction enzymes
cleavage site can be up to 1000 bp away from the recognition site
Type I restriction enzymes
closer cleavage sites, usually up to 20 to 30 bp away from the recognition site
Type III
cut which leaves single-stranded 3’ or a 5’ overhang, that easily re-attach to other ends (sticky ends)
staggered cut
cut that doesn’t leave an overhang
blunt double-stranded cut
what is needed for a molecule to be cloned
a vector and an insert
What should an ideal vector be able to do?
replicate autonomously and be able to amplify insert DNA up to 10 kb
what does a vector generally contain?
-selectable marker
-origin of replication
-restriction site
Explain the cloning strategy:
-restriction enzyme cuts vector and insert
-vector and insert are combined
-complementary overhangs are fused together with T4 DNA ligase
-ligated molecules are used to transform E.coli
the process by which DNA is introduced into a host cell
transformation
describe the transformation process:
-competent E.coli take up outside DNA
-bacteria placed on antibiotic plates to see which ones took up a plasmid
-colonies form
cluster of identical plasmid-containing bacteria
colony
cloning that relies on the hybridization of the complementary base pairs Adenine and Thymine
TOPO cloning (Topoisomerase based cloning)
describe the process of TOPO cloning:
-Taq polymerase leaves a single A overhang on the 3’ end of PCR products or insert
-Thymine comes from a pre-cut , linear cloning ready TOPO vector that has a DNA topoisomerase I fused to the 3’ end
-topoisomerase acts as a ligase & joins A&T together
-
describe the process of TOPO cloning:
-Taq polymerase leaves a single A overhang on the 3’ end of PCR products or insert
-Thymine comes from a pre-cut , linear cloning ready TOPO vector that has a DNA topoisomerase I fused to the 3’ end
-topoisomerase acts as a ligase & joins A&T together
screening technique that allows for quick and easy detection of successful ligations in vector-based gene cloning
blue/white screen
describe blue/white screening for positive transformants:
-DNA is ligated into a vector
-vector is transformed into competent cells that are grow in the presence of X-gal
-success=white colony; unsuccessful=blue
what operon does blue/white screening utilize
lac
How is the presence of an active B-galactosidase detected by X-gal within the agar plate?
X-gal is cleaved by B-galactosidase to form a bright blue insoluble pigment
what do the blue and white colonies contain
-blue contain the vector without an insert because the B-gal cleaved Xgal into a blue compund
-white remains colorless bc the inactivated B-gal did not cleave X-gal
what vectors are used to clone larger inserts, express cloned genes in cells, investigate properties of a promoter or create various fusion proteins?
expression vectors
introduction of gene of interest into the host cell and aids in the analysis of the foreign gene via relevant protein product expression
dual function
-a naturally fluorescent protein that, when excited with one wavelength of light, emits fluorescence in another wavelength
-used to visualize patterns of gene expression
Green fluorescent protein (GFP)
a protein of interest is fused to GFP and can thus be visualized in living tissues
fusion proteins
gel electrophoresis
separates DNA fragments by size, using an electric current to cause DNA to migrate toward a positive charge; smaller fragments migrate faster
electrophoresis is done by preparing a small slab of gel in an electrically conductive mildly basic buffer; negatively charged DNA moves by electric current (smaller moves faster)
Agarose Gel Electrophoresis
permits the exponential amplification of a desired sequence
PCR
uses reverse transcriptase to convert RNA to DNA for use in a PCR
RT-PCR
detects the products of PCR amplification during their synthesis and is more sensitive and quantitative than conventional PCR
Real-time or quantitative PCR (qPCR)
PCR depends on the use of _______________________ that can withstand multiple cycles of template denaturation
thermostable DNA polymerases
What are the components of PCR?
-DNA template
-DNA polymerase
-Primers
-Nucleotides (dNTPs)
What are the repeated steps of PCR?
Denaturation, annealing, extention
the two strands of DNA are separated; normally done at 94 C
Denaturation
the primers bind to the DNA; normally done at 55 C
Annealing
using the DNA template, the polymerase adds the dNTPs to the primer and causes the extension of the prime; normally done at 72 C
Extension
what does repeating the pcr step lead to
N(thermal cycles) exponential increase (2N) allowing for phenomenal levels of amplification
-combines the effects of reverse transcription and quantitative PCR to amplify and detect specific targets
-variety of applications including quantifying gene expression levels, validating RNAi, and detecting pathogen
RT-qPCR
allows collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step
-detection based on fluorescence technology
Real-time PCR
-detection of nucleic acid to complementary sequences
-involves the bonding of a probe to a target seqence
hybridization
short, complementary nucleic acid strand
-generally labeled with a radioisotope or fluorescent molecule
probe
a positive hybridization signal is obtained when _____________ occurs between the probe and the target sequence
complementary base pairing
-allows the detection and localization of viral nucleic acid in tissue sections or cytological specimens using labelled nucleic acid probes with complementary sequences to the target viral nucleic acid
in situ hybridization (ISH)
uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope
fluorescence in situ hybridization (FISH)
commonly used for nucleic acid detection following the hybridization of a radioactive probe to filter bound DNA or RNA
blotting techniques
hybridization of a probe to filter bound DNA
southern hybridization
hybridization of a probe to filter bound RNA
Northern hybridization
involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridization with a labeled probe
Southern Blotting
involves determination of nucleotide bases A,T,G and C in the DNA
DNA sequencing
The classic method of DNA sequencing called _____________________________________ has been significantly used since its discovery by Frederick Sanger and colleagues in 1977.
dideoxy sequencing or Chain termination reaction
method that requires many identical copies of DNA, an oligonucleotide primer that is complementary to a short stretch of the DNA, DNA polymerase, deoxynucleotides, and dideoxynucleotides (ddNTPS)
Sanger Sequencing
why is it called the chain termination reaction
ddNTPs terminate DNA synthesis at particular nucleotides
modified nucleotides that can be incorporated into the growing DNA strand but lack the 3’ hydroxyl group needed to attach the next nucleotide
ddNTPs
what is the difference between chain-termination PCR and standard PCR
ddNTPs are added in chainterm and causes extension to cease
what is the result of chain-termination PCR
millions to billions of oligonucleotide copies of the DNA sequence of interest, terminated at random lengths (n) by 5’-ddNTPS
what side of the electrode is DNA pulled to
positive because it is negative
shows the fluorescent peak of each nucleotide along the length of the template
chromatogram
what type of electrophoresis is used with Sanger sequencing
capillary gel
-allows detection of specific protein-DNA interactions in vivo and mapping of all the protein-binding sites for a given protein across the entire genome
-used to identify the relative abundance of a specific protein or specific protein modification at a certain region in the genome
Chromatin Immunoprecipitation (ChIP)
how are antibodies utilized in ChIP
antibodies selectively recognize and bind proteins, including histones, histone modifications, transcription factors, and cofactors to provide information about chromatin states and gene transcription
describe the process of ChIP
-protein and DNA are crosslinked
-chromatin broken into small fragments
-antibody used to immunoprecipitate protein of interest
-DNA then purified and analyzed by PCR, sequencing, or by labeling the DNA and applying to a tiling array to detect genome-wide interactions
