Chapter 20 Recombinant DNA Technology

Recombinant DNA technology is the use of __________ techniques to isolate and manipulate DNA.
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Recombinant DNA technology is the use of __________ techniques to isolate and manipulate DNA.
in vitro
Gene cloning is the process of isolating and making many copies of a gene in vitro or in vivo.
...
Molecular geneticist's toolbox:
- restriction enzymes
- DNA polymerase
- dNTPs
- ligase
- cloning vectors
- restriction enzymes = cut DNA
- DNA polymerase = synthesis of DNA
- dNTPs = A,T G,C
- ligase = joins DNA together
- cloning vectors = (CV), harbor gene of interest for cloning
gene cloning in vivo

(3 steps)
1. Isolate DNA (ex: chromosomes) from an organism or collection of cells.

2. Cut DNA and CV with same RE. Insert (ligate) DNA fragments into CV to make a
recombinant DNA molecule.

3. Transform host cell (ex: E. coli) with recombinant CV. Host cell and recombinant CV
replicate multiple times to generate identical copies of the GOI.
restriction site sequences are usually
palindromic
restriction enzymes can cut two ways :

symmetrical or asymmetrical
symmetrical produce blunt ends

asymmetrical cuts produce sticky ends
Formation of a Recombinant DNA Molecule
involves restriction enzymes and DNA ligase
Cloning Vectors:

Cloning Vectors
E. coli plasmid CVs must have:

1.

2.

3.

4.
Cloning Vectors
E. coli plasmid CVs must have:

1. ori to replicate within host cell

2. multiple cloning sit/MCS (poly linker)

3. antibiotic resistance gene

4. lacZ gene spanning multiple cloning site
Blue white screening (4 steps)
1. Cut CV with RE.

2. Cut chromosomal DNA with same RE.

3. Add ligase to create recombinant CV.

4. Transform bacteria with recombinant CV and
plate on agar containing ampicillin and X-gal
Self-ligation can be prevented by ___________
alkaline
phosphatase.
Artificial transformation via: cold CaCl2 and heat shock and electroporationsynthesis of complementary DNA (cDNA) cDNA is used for cloning genes starting with mRNA vs. Chromosomal DNA. This procedure applies only if you are interested in...cloning the coding sequence of a gene instead of all of it (introns and exons), applies to eukaryotes. basically, converting mRNA into cDNA then cloning the cDNAGene Cloning in VitroPolymerase Chain Reaction Need: target DNA, two primers, Taq polymerase, dNTPs, thermal cycler Procedure : __________________________Procedure: 1. Denature at 95oC 2. Anneal primers at 45-68oC 3. Extend primers at 72oC. 4. Repeat 20-40x.Reverse Transcriptase-PCR RT-PCR is used to detect the presence of a ________________specific mRNA.Reverse Transcriptase-PCR RT-PCR is used to detect the presence of a specific mRNA. Procedure: 1. _________________ 2. _________________1.) make cDNA from mRNA by RT 2.) amplify cDNA by PCRPCR and RT PCR are end point semiquantitative meaningit cant tell us the exact levels of DNA that have been amplified. just saying presence ofReal-Time PCR qPCR is used to detect the quantity of DNA present or RNA expressed. Procedure: 1. 2.1.) synthesize cDNA from mRNA via RT 2.) PCR with either a SYBR green or taqman probeGene Targeting OverviewGene targeting is the process of altering a genetic sequence and observing the consequence _______________in vivo.1. _______________ (transgenic) organisms are used to study the function of a gene when it is over-expressed in vivo. 2. _______________ organisms are used to study the function of a gene when it is under- expressed in vivo.1. Knock-in (transgenic) organisms are used to study the function of a gene when it is over-expressed in vivo. 2. Knockout organisms are used to study the function of a gene when it is under- expressed in vivo.Transgenic Mice Procedure: 1. 2. 3. 4.1.)Inject transgene (TG) into pronucleus of fertilized egg. TG will randomly insert into chromosome via nonhomologous recombination 2.) Implant embryos into a foster mother 3.) embryos will express TG in all cells 4.) breed to produce TG miceKO (knock out) mice