BIMS 320 chapter 15: Recombinant DNA tech

Know the definition and basic procedure of recombinant DNA technology.

- set of techniques for amplifying, maintaining, and manipulating DNA sequences in vivo and in vitro

- basic procedure:

- donor DNA and vector (DNA molecules that accept DNA fragments) both fragmented with restriction enzymes

- DNA fragments joined together with vector

- recombinant DNA molecules added to host cell to produce copies which can be recovered = CLONES

What is a clone?

- recovered copies of a recombinant DNA molecule

- used to study the structure and orientation of DNA

Restriction enzymes bind to what?

Restriction Site = specific recognition sequence of the DNA

What is the symmetry that recognition sequences exhibit and how long can they be?

- palindrome: a sequence that when flipped is identical to the complementary sequence

- mostly 4-6 nucleotides long

- sometimes 8+ nucleotides

Know the difference between a sticky and blunt end.

- ends of segments produced by restriction enzyme cuts

- sticky ends: one strand of DNA longer and hangs over the other

- blunt ends: no over hangs

Be able to identify the restriction site for HindIII, EcoRI, BamHI, and AluI and what fragment is produced (blunt/sticky).

- HindIII: A/AGCTT sticky

- EcoRI: G/AATTC sticky

- BamHI: G/GATCC sticky

- AluI: AG/CT blunt

Understand the procedure for creating Recombinant DNA with restriction enzymes.

- OG DNA and vector cleaved with restriction enzyme

- vector and donor dna fragment ends combine via base pairing

- DNA ligase seals phosphodiester backbone and actually covalently links two strands

What is a restriction map and how can they be used?

- maps of DNA sequences

- provide info for future experiments like subcloning (cloning subsections of DNA sequence)

What is a plasmid

extrachromosomal, double stranded, circular DNA molecule that replicates independently from chromosomes

How can selectable markers identify recombinant DNA and what are examples of the genes that are used?

- genes that provide resistance to antibiotics are effective selectable markers

- Ampicillin presence - used to determine if the bacteria contains the vector (ampicillin resistant gene)

- lacZ functionality - used to determine if the DNA has been cloned into the MCS

How is transformation achieved?

1. using calcium ions and brief heat shock to pulse DNA into cells

2. Electroporation: uses brief pulse of electricity to move DNA into bacterial cells

What is the result of the blue-white screening mechanism and the components it uses?

- used to ID cells containing recombinant and nonrecombinant DNA

- plasmid contains lacZ gene and ampicillin

- DNA fragments added to multiple cloning site of plasmid and disrupts plasmid's lacZ gene

- Recombinant plasmids have disrupted lacZ gene, can't metabolize X-gal, form white colony

Non-recombinant plasmids have functional lacZ gene, metabolize X-gal, form blue colony

Mechanism:

- restriction enzymes cut DNA

- insert into vector

- ligate

- plate it

- ID desired clone

What are the other types of vectors?

- Phage vectors - have genetically modded strains of bacteriophage (λ)

- eukaryotic expression vectors - used in yeast or tissue culture cells in situations where bacteria cant produce functional prods of a transgene

- BACs

- YACs

What is a BAC/YAC and what is the insert size capacity of a BAC?

- both are vectors; BACs are preferred

- Bacterial artificial chromosomes (BACs): insert size capacity 100-200 kb

- Yeast artificial chromosomes (YACs): used to clone DNA sequences in yeast cells

___________ and soil bacterium can be used for introducing genes in plants.

Ti plasmid (prokaryotic expression gene)

When are eukaryotic expression vectors used?

- in yeast or tissue culture cells in situations where bacteria cant produce functional products of a transgene

- designed to ensure mRNA expression of cloned gene - to produce many copies of encoded protein in host cell

What are the differences between DNA, genomic, and cDNA libraries?

- DNA library: collection of cloned fragments of DNA, usually from 1 source

- Genomic libraries: derived from genomic DNA of an organism; contains many overlapping frags of the genome

- Complementary DNA (cDNA) libraries: contains complementary DNA copies made from mRNAs isolated from cultured cells or tissues

Genomic libraries contain all of the DNA. T/F

True

What is a probe?

- Any DNA or RNA sequence complementary to target gene of sequence being ID'd

- must be labeled/tagged

- used to screen library and recover clones of specific gene

PCR overview + steps w/ temperatures

- Polymerase chain reaction (PCR): rapid method of DNA cloning, copies specific DNA sequence via in vitro rxns, just needs small quantities of target sequence (source examples: dried blood, semen, hair)

- Steps:

1. Denaturation (92-95 C) - double stranded DNA denatured into single strands

2. Hybridization/Annealing (45-65 C) - primers bind to ssDNA

3. Extension (65-75 C) - DNA polymerase synthesizes DNA strands

- each cycle results in amplification - products of previous cycle serve as templates for next

Difference between RT-PCR and qPCR.

- Reverse transcriptions PCR (RT_PCR): way to study gene expression (reverse transcriptase used to generate cDNA)

- Quantitative real-time PCR (qPCR): lets ppl quantify amplification reactions as they occur in real time

What is gene targeting and gene knockout?

- gene targeting: target specific allele, locus, or base sequence and learn its function on gene of interest

- gene knockout: loss of function mutation, eliminate specific genes and see what happens

True/False: Insulin was among the first human genes to be expressed in E. coli.

true

Knockout mice used ________ cells to be injected into a blastocyst.

embryonic stem (ES)

What transgenic model system did we use as an example in class and what was it trying to achieve?

a) Color of final mice and gene expression. Brown v. black (+/+, +/-, -/-)

- we used the CFTR model

- trying to generate knockout mice from ES cells

- blastocyst-stage mammalian embryo has ES cells; ES cells isolated and grown in culture

- DNA introduced & cells transferred to media that selects for + marker and against - marker

- transformed cells added back to blastocyst embryo of dif mouse

- blastocyst implanted into surroate demale and embyo develops into mosaid mouse w/ some tissues derived from transformed ES cell

- some gametes of that mouse might be from transformed cells so offspring will be heterozygous for the transgene

- brown mouse +/-, black mouse +/+, homo mouse prod from hetero -/-

What is the best approach for genome editing?

CRISPR-Cas

germinal gene therapy has been performed in humans. T/F?

false

germinal gene therapy: targets cells of germ line which give rise to gametes; so progeny of treated individual gets transgene

True/False: Crips-Cas9 uses sgRNA and dsDNA to break repair mechanisms.

false; only sgRNA

Gene editing with CRISPR-Cas9 has been used to delete mutant exon 23 from a mouse model of Duchenne Muscular Dystrophy (DMD) loss of function alleles in _________ gene.

Dystrophin

What is totipotent?

- way to describe most plant cells

- means that under appropriate conditions a normal plant can be regenerated from a single isolated plant cell