CH. 15 Recombination DNA Technology

Difference between RT-PCR and qPCR

- Reverse transcription PCR (RT-PCR) methodology for studying gene expression (mRNA production by cells or tissues) -Quantitative real-time PCR (qPCR) real-time PCR allows researchers to quantify amplification reactions as they occr in real time

What transgenic model system did we use as an example in class and what was it trying to achieve? (slides46-52) . Color of final mice and gene expression. Brown v. black and knockout or normal

- creating a loss -of-function CFTR (Cystic Fibrosis Transmemebrane Conductance Regulator) Allele in Mice Through Holologus Recombination - create chimeras -offspring mouse will be homozygous for the transgene

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, disrupt or eliminatre specific gene/genes and see "what happens." Knockout (KO) mice have revolutionized research.

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

- palindrome -mostly 4-6 nucleotides long (sometimes more than 8)

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

- provides information for further experiments, such as subcloning, cutting of DNA into smaller fragments in order to separatelt clone subsections of the sequence

what is the result of the blue-white screening mechanism and the components it uses? (slide 20-21) a.

-Blue-white selection: used to identify cells containing recombninant and nonrecombinant DNA -Agar plates contain X-gal - when x-gal is cleaved by enzymes, it turns BLUE -Bacterial cells with functional lacZ gene carrying a nonrecombinant plasmid= BLUE -bacterial cells with recombinant plasmid= WHITE

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

-DNA library: is a collection of cloned fragments of DNA, usually from a single source - genomic libraries: libraries developed from the genomic DNA of an organism -cDNA libraries: derived by mRNA and are called complementary DNA (cDNA)

Know the defentition and basic procedure of recombinant DNA technology

-Recombinant DNA technology is a set of techniques for amplifying, maintaining, and manipulating DNA sequences in vitro and in vivo procedures: 1. generating specific DNA fragments using restriction enzymes 2. joining these fragments with a vector 3. transferring the recombinant DNA molecules to a host cell to produce many copies that can be recovered from the host cell

What are the other types of vectors?

1. phage vectors: 2. Bacterial artificial chromosomes (BACs) 3. Yeast artificial chromosomes (YACs) 4. Eukaryotic Expression vectors: engineered to express a gene 4. Ti plasmid and soil bacterium

The amino acid sequence of insulin was determined in the ________***

1950s

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 the best approach for genome editing?

CRISPR-Cas9

True/False: germinal gene therapy has been performed in humans

FASLE -has NOT been attempted on humans

restrictions enzymes bind to what?

DNA at the restriction site

be able to identify the restriction site fot HindIII, EcoRI, BamHI, and AluI and what fragment is produced (blunt/sticky) (slide 9)

HindIII: sticky ends EcoRI: sticky ends BamHI: blunt ends Alul: sticky ends

T/F Genomic libraries contain all of the DNA.

TRUE

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

TRUE

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

TRUE

What is a probe?

any DNA or RNA sequence complementary to target gene of sequence being identified

What is totipotent?

immature or steam cell capable of giving rise to any cell type

When are eukaryotic expression vectors used?

must be used in yeast or tissue culture cells in situations where bacteria cannot produce functional products of a transgene

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

-bacterial artifiical chromosomes and yeast artifiical chromosomes - BACs have a insert size capacity of 100-200 kb

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

-by cutting the vector within the selectable marker will interrupt the gene, and allow for identification of recombinants. -ampicillin is used to determine if the bacvteria contain the vector (ampicillin resistant gene) - the lacZ gene is used to determine if the DNA has been cloned into the MCS

what is a clone?

-recovered DNA copies in recombinant DNA molecules - they can be used to study the structure and orientation of the DNA

understand the procedure for creating Recombinant DNA with restriction enzymes. (slide 10-11)

-restriction enzyme identify in Escherichia coil: ~ produces DNA fragments with cohesive ends (sticky ends) ~ ssDNA fragments from different sources can anneal (stick together) by hydrogen bonding

know the difference between a sticky and blunt end

-sticky ends: single-stranded segments produced by some restriction enzyme cuts (can base-pair with complementary sequences) -blunt ends: no single-stranded overhangs

how is transformation achieved?

1. Using calcium ions and brief heat shock to pulse DNA into cells 2. Electroporatin: which uses a brief but high-intensity pulse of electricity to move DNA into bacterial cells

_____ and ______ are two important tools used to construct and amplify DNA molecules?

1. Restriction enzymes: DNA cutting enzymes 2. Cloning vectors

qPCR can evaluate gene expression when correlated to what output?**

RFU (relative fluorence units) to cycles. cMSCs?

Steps in PCR with temperatures (also review from older material; slide 36)

Three steps of PCR: 1. Denaturation: dsDNA is denatured into single strands 2. Hybridization/Annealing: Primers bind to ssDNA-- starting point for DNA polymerase to synthesize new DNA strands 3. Extension: DNA polymerase synthesizes DNA strands -each cycle results in amplification-- products of previous cycle serve as templates for each subsequent cycle (chain reaction)

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

Ti plasmid

what is a plasmid?

a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. Plasmids are much used in the laboratory manipulation of genes.

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

embryonic stem cells

TAMU was the first intuition to clone 3 species, and some species discussed in class included ?.***

horses, pigs,goats, cattle and deer