Chapter 10 — Recombinant DNA: Gene Isolation and Manipulation

blotting

(DNA must be denatured first which allows it to stick to the membrane) paper towels + membrane + agarose gel (post -electrophoresis) + sponge + salt solution ...hybridize membrane with probe in a ziplock bag ...wash away unbound probe, bound probe attaches to complementary seq.

genetic engineering in mice

(a) ectopic insertions : transgenes inserted randomly in the genome (usually as multi-copy arrays) (b) targeted insertion/gene targeting : transgene inserted into a location occupied by a homologous sequence in the genome (transgene replaces its normal homologous counterpart)

Modes of delivering recombinant DNA into bacterial cells

(a) plasmids, BACs : circle + bacterial cells --> transformation --> colony (b) fosmids : phage + bacterial cells --> transduction --> colony (c) bacteriophages : phage + bacterial cells --> infection --> lysis, plaque

finding a specific clone of interest :

(a) those that recognize a specific sequence of DNA or RNA (oligo sequences i think) (b) using antibodies that recognize a specific protein

PCR : Primer extension

*heat* again to 72 degrees C, taq polymerase uses dNTPs (dATP, dTTP, dGTP, dCTP) to extend the strand, thermostable temperature for extension at 72 degrees C in vitro: use thermophillic Taq, not a regular polymerase taken from the bacteria thermus aquaticus because it can withstand high heat

formation of recombinant DNA molecule

- 2 sources of DNA - cleaved by a restriction enzyme - hybridization with ligase

gel electrophoresis :

- DNA migrates to anode (+) end - bands visualize by staining DNA with ethidium bromide - DNA fragments of distinct sizes form bands - smaller go farther

ddNTPs

- modified nucleotide *key* ingredient in the Sanger sequencing technique because of its ability to *block continued DNA synthesis* -lacks the 3' and 2' hydroxyl group -DNA polymerase needs to catalyze a reaction between the 3' hydroxyl group of the last nucleotide and the 5' phosphate group of the next nucleotide to be added therefore it is unable to and addition fails

choosing your cloning vector :

- must be small for convenient manipulation - integrity of DNA insert : do they need to be able to replicate in living cells? or do you only need one copy - must have convenient restriction sites - must have a way to identify and recover the recombinant molecule

plasmid vectors

- small, circular DNA molecules - replicate independent of the bacterial chromosome (ori: start site of replication) - restriction site (e.g.: polylinker, insert the DNA) - carry a gene for drug resistance (e.g.: ampR) in order to select for bacterial cells transformed by plasmids - carry a gene to distinguish plasmids with and without DNA inserts (e.g. lacZ gene + lac promoter region)

Why use DNA primers versus mRNA ?

- the stability of DNA - use of enzymes that react with DNA and not RNA

how can we use crispr/cas9 for genetic engineering?

-sgRNA to target any specific sequence as long as we also have the associated PAM sequence and we can cut anything we want -2 different sgRNA will help us cut out a region of DNA on the same genome -this allows us to selectively "knock out" OR "knock IN" any region of the genome -also used to switch on or off the gene by having the cas9 nucleoprotein simply sit on a targetted gene to prevent transcription factors from binding to it

recombinant DNA techniques:

1. Cleavage of DNA at specific sites - restriction nucleases 2. DNA ligation - to design and construct DNA molecules not found in nature 3. DNA cloning - cloning vectors with the polymerase chain reaction in which a portion of DNA is repeatedly copied to generate many billions of identical molecules 4. Nucleic acid hybridization - find a specific sequence of DNA or RNA with great accuracy and sensitivity on the basis of its ability to selectively bind a complex nucleic acid sequence 5. DNA sequencing - Rapid determination of the sequence of nucleotides of any DNA (even entire genomes) 6. Microarrays

PCR ingredients:

1. Primers 2. dNTPs 3. DNA polymerase (taq) 4. Target DNA 5. Mg2+ (not too much, not too little - catalyzes taq polymerase activity)

Amplification of donor DNA inside a bacterial cell

1. donor DNA is cut by a restriction enzyme 2. restriction fragments are ligated into plasmid insert to form recombinant DNA molecule 3. recombinant DNA molecule is taken up by bacterial cell through transformation 4. inside the cell, recombinant DNA is replicated and amplifies 5. cell division = clones with donor fragment 6. recover the recombinant DNA molecule

sources of donor DNA :

1. entire genome : restriction enzyme digest 2. PCR products 3. cDNA : DNA copy of mRNA

producing PCR products with sticky ends :

1. first round of PCR : 3' end of primers anneal to target sequence. there is a 5' end that is left untouched by primers 2. second round of PCR : 3' end of primers attaches to an amplified piece of DNA that has previous primer on the 5' end that was previously left untouched 3. third round of PCR : products both ends contain matching primer ends that book end the product. these complementary, palindromic strands are then digested by EcoRI or another restriction enzyme and become new sticky ends

use of a plasmid vector :

1. foreign DNA and plasmid vector cut with restriction enzyme 2. transform bacteria 3. placed on plate with ampicillin X-Gal and IPTG 4. (a) with insert, lacZ- = white colonies (b) no insert, lacZ+ = blue colonies

use of bacteriophage vector :

1. harbors DNA as package inside phage head 2. central part of phage DNA is not needed for replication or packaging so it can be cut out and discarded by restriction enzyme 3. the deleted central part of phage is then replaced by inserts of donor DNA

producing cDNA molecules with sticky ends :

1. initially we have double-stranded cDNA with blunt ends 2. ligate oligonucleotide linkers containing EcoRI's palindromic sequence 3. cut with EcoRI = sticky ends! 4. ligate into a vector that has also been cut with EcoRI

ectopic insertion

1. inserted directly into nucleus of eggs 2. injected eggs inserted into oviduct, some develop into baby mice cons: expression pattern of genes may be abnormal (position effect) and mutations can occur

cloning vectors :

1. plasmid vectors 2. bacteriophage vectors 3. fosmids 4. bacterial artificial chromosome (BAC) F- plasmids

using probe for finding protein :

1. requires an *expression library* made by using expression vectors that will produce a protein 2. requires an *antibody* to the specific protein product of the gene of interest -place membrane over protein colonies - protein fusion onto membrane - primary antibody binds to protein - second labeled antibody attaches to first antibody and locates visibly the protein of interest

using a probe for finding DNA/RNA seq. :

1. transfer bacterial cells containing a fosmid library to an absorbent membrane by laying a nitrocellulose membrane on the surface of the colonies 2. lyse the colonies (probe is single stranded), label with either flourescent dye or radioactive isotope 3. location will visibily appear on the membrane 4. isolate the located clone 5. grow the isolated colony and amplify the desired gene

methods of introducing a transgene :

1. transformation 2. projectile gun 3. mechanical injection 4. virus

Where does the DNA to make a probe come from?

?

Cas9 nuclease

A bacterial endonuclease/nucleoprotein that cuts double-stranded DNA at a location determined by a segment of guide RNA. -only activated when tracrRNA and crRNA ( --> single guide RNA) bind together via overlap for cas9 to work properly -has the channel for DNA to fit into -scans DNA looking for a specific sequence -PAM sequence is the final check to see if its a match -RNA binds to the strand and opens the double helix -bacteriophages DNA gets cut very close to the PAM site -bacteriophage essentially dies after cutting

Biotechnology

A form of technology that uses living organisms, usually genes, to modify products, to make or modify plants and animals, or to develop other microorganisms for specific purposes.

gene knockout

A gene may be inactivated by substituting an inactive gene for the normal gene carried in embryonic stem cells (ES) cultures stage 1: produce ES cells for gene knockout stage 2: transfer ES cells into mice embryos

the constructing of a human genomic DNA library :

A genomic library is usually stored as a set of bacteria, each bacterium carrying a different fragment of human DNA

probe

A single-stranded DNA or RNA fragment used in genetic engineering to search for a particular gene or other DNA sequence. The probe has a base sequence complementary to the target sequence and will thus attach to it by base pairing.

genetic engineering

A technology that includes the process of manipulating or altering the genetic material of a cell resulting in desirable functions or outcomes that would not occur naturally.

lacZ encodes the enzyme _____________.

B-galactosidase if lacZ includes an insert, no B-galactosidase = white colonies, X-gal not cleaved by enzyme if lacZ is not disrupted by an insert, = functional B-galactosidase, X-gal cleaved by enzyme

southern blotting

DNA analysis

Sanger sequencing method

Divided into 4 samples (ddA, T, G, C) Label with radioactive/dye oligo at 3' end Mix with taq, dNTPs, ddNTP and incubate run on gel --> frags will terminate at different lengths

What makes Taq adapted to withstand high heat?

It's protein folding structure LOW entropy required for it to fold

Whats the benefit of different species of bacteria making different restriction nucleases?

Protects them from viruses by degrading incoming viral DNA

northern blotting

RNA analysis

dideoxy chain termination method

Short DNA fragments can be sequenced by this. Uses dideoxyribonucleotides that are modified nucleotides that attach to synthesized DNA strands of different lengths. Each ddNTP is tagged with flourescent label.

Why are Magnesium cations important in PCR?

Stabilizes the binding of polymerase concentration of magnesium affects the binding of taq. too much: too stable, binds in wrong places, gaps and non-specific products. too little: barely any product at all

CRISPR/Cas9

a revolutionary gene editing technique derived from the immune system of simple prokaryotes basically and efficient genetic scissors unlike transgenetic techniques which can leave foreign DNA in the genome features: -highly specific -tightly regulated -highly efficient

the polymerase chain reaction is :

an exponential amplification of DNA, Kary Mullis discovered it on LSD in the 70s

Hpal cuts DNA into ___________

blunt ends

Steps of PCR :

denaturation, annealing, extension

gene targetting

enables us to eliminate or modify the function encoded by a gene 1. gene replacing 2. gene knockout

cloning DNA fragments with blunt ends :

even though they can be joined to the vector plasmid with ligase alone, this is an inefficient reaction because the blunt ends cannot stick together with hydrogen bonds

fosmids and BACs are cloning vectors that carry ___________ inserts

large (80-90% of vector)

Why is genomic DNA not a good starting point if the goal is to isolate and analyze genes that encode proteins?

mRNA is a preferred starting point in gene isolation ? the enzymatic conversion of mRNA into cDNA allows for the isolation of a gene copy without introns

cDNA synthesis

making a complementary DNA sequence of your mRNA with reverse transcriptase in cell: -transcription from DNA to mRNA -introns removed from mRNA, polyA tail added to 3' end -oligo dT primer binds to polyA tail -reverse transcriptase extends primer with dNTPs moving toward 5' creating the cDNA from mRNA template strand -strand loops back onto itself, mRNA is removed -DNA polymerase copies cDNA strand, creating double-stranded cDNA -nick hairpin

How do the bacterial enzymes know not to cut up the host DNA?

methylation adding a methyl group to the end of a DNA segment prevents cleaving of their own DNA

genetic engineering in plants

most commonly used: Ti plasmid vector (Tumor-Inducing) derived from natural plasmid from soil bacterium, gauses crown gall disease/tumors *T-DNA*: region of Ti plasmid that inserts into host plant, the genes whose products catalyze the transfer of T-DNA separate from the region on T-DNA of the Ti plasmid themselves (this is the plants natural behavior) - because of the natural behavior, scientists replaced the tumor-causing genes that were previously present between the T-DNA borders with whatever DNA they wanted

gene replacing

mutant allele can be replaced by substituting wild-type allele for the mutant in its normal chromosomal location method avoids both the position effect and DNA mutations/rearrangements associated with ectopic insertion because a single gene is inserted into its normal chromosomal environment

recovery of amplified recombinant molecules :

phage particles : easily obtained by collecting phage lysate and isolating the DNA they contain plasmids, fosmids, BACs : bacteria are first chemically, or mechanically broken apart by techniques that select the recombinant plasmid by distinguishing through shape or size (e.g.: electrophoresis, centrifugation)

PCR : Primer annealing

primers bind to their complementary sequence on the template strand (both strands, one primer on each) creating H bonds and new base pair on the 3' end, bookending the segment of interest, *cooled* to 55 degrees C

homologous recombination

process that results in genetic exchange between homologous DNA from two different sources

western blotting

protein analysis

________________________ cut large DNA molecules into fragments at ______________, ______________ sites.

restriction nucleases; specific, palindromic

PCR : DNA denaturation

rid the duplex to get single strands with *heating* at high temperature of 95 degrees C to destroy hydrogen bonds and expose the DNA nucleotides for DNA polymerase and PCR DNA primers to access them

How does DNA polymerase know where to stop?

segment of DNA ends or reaches the other primer's start location

Pstl cuts DNA into ___________ with ____________

sticky ends, 3' overhangs

EcoRI cuts DNA into ___________ with ____________

sticky ends, 5' overhangs

HindIII cuts DNA into ___________ with ____________

sticky ends, 5' overhangs

Homologous

term used to refer to chromosomes that each have a corresponding chromosome from the opposite-sex parent

recombinant DNA :

the joining of DNA molecules, usually from different biological sources that are not found together in nature

gene therapy

the transplantation of normal genes into cells in place of missing or defective ones in order to correct genetic disorders. transformed cells infused back into patients cons: insertion of viral vector can inactivate important genes

genetic engineering in animals

transgenic DNA is *injected* directly as plasmids or fosmids

YIPs

yeast integrated plasmids: derivatives of bacterial plasmids into which the yeast DNA of interest has been inserted when transformed by yeast cells, these plasmids insert into yeast chromosomes by *homologous recombination*

Why don't we always use PCR?

you must know the sequence you want to amplify with PCR in order to make/use the right primer to cleave in the right spot