BiotechnologyCh 20

20.1 DNA cloning Making copies of segments of DNA Gene cloning – making multiple copies

of a gene Why? To make many copies of a gene

(amplification) To produce a protein product

Cloning & bacteria Plasmids are frequently used in

cloning genes Gene of interest is inserted into

plasmid: Recombinant DNA – DNA from 2

different sources Plasmid is inserted into bacteria,

bacteria divide, producing copies of genes

Recombinant DNA DNA from two sources is combined http://www.youtube.com/watch?v=

8rXizmLjegI

Cloning a Gene1. Isolate vector and gene of interest

Determine vector – molecule that will carry foreign DNA, and gene of interest

Vector may have particular genes to aid in recognition of of cell clones vector - bacterial plasmid

Has ampR – ampicillin resistance gene

Has lacZ gene – catalyzes hydrolysis of lactose sugar – at restriction site, so the enzyme cuts in middle of gene

Gene - human gene of interest

Restriction Enzymes Protects bacteria

by cutting up foreign DNA

Work on specific sequences of DNA, usually symmetrical

Result in “sticky ends”

2. Insert gene into vector

The same restriction enzyme is used to digest the plasmid (only one recognition site), and human DNA Result- human DNA is cut into many

fragments – one is the correct one. Cut plasmids and DNA fragments are

mixed together. Sticky ends join through complementary base pairing. DNA ligase is used to form phosphodiester bonds to join DNA molecules.

Making recombinant DNA

3. Introduce cloning vector into cells

Bacterial cells take in recombinant plasmids through transformation, taking in DNA from surrounding solution

4. Cloning of cells

Bacterial cells are plated out onto nutrient medium with ampicillin and X-gal sugar added

Need to determine which bacterial cells contain recombinant plasmids Only bacteria with recomb. plasmids will grow on

medium with ampicillin, because of ampR gene Bacteria with the intact lacZ gene turn blue with

hydrolysis of X-gal, but bacteria with recomb plasmids cannot process X-gal sugar, so are white

Cloning a gene - video http://highered.mcgraw-hill.com/olcwe

b/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Steps%20in%20Cloning%20a%20Gene

5. Identify cell clones with gene of interest

Need to find bacteria with plasmids that contain gene of interest, vs. other human DNA fragments

Use nucleic acid probe – short strand of DNA or RNA that is complementary to part of gene of interest

DNA is denatured, and then radioactive or fluorescent probe is added

Genomic Libraries A collection of many clones A complete set of plasmid-containing

cell clones When no single gene is target,

genome broken into fragments, each gets recombined into a plasmid

Bacterial artificial chromosome – larger than plasmids, hold more genes

Complementary DNA - cDNA Eukaryotic DNA from its original

source includes introns To get around this problem, start

with a fully processed mRNA strand Use reverse transcriptase to

synthesize double stranded DNA Can build a cDNA library

Nucleic Acid Hybridization

Can be used to label particular bands of DNA

Synthesized radioactively labeled RNA hydrogen bonds with target complementary DNA

Cloning & expressing eukaryotic genes

Problems due to differences in how prokaryotic & eukaryotic cells express genes

Promoter- use an expression vector with a promoter sequence upstream of insertion site, so host cell recognizes it and will express gene that follows

Introns – find processed mRNA, use reverse transcriptase to make complementary DNA (cDNA) that can be used in bacteria

Yeast – hosts for eukaryotic cloning Advantages: Single- celled fungi, easy to grow Have plasmids (unusual for eukaryotes)

Eukaryotic host cells can modify proteins after translation, bacteria can’t do this

PCR – Polymerase Chain Reaction Making copies of DNA Uses heating & cooling cycles to: 1) denature – separate DNA strands 2) anneal - bind primers at ends 3) extension -synthesize DNA with DNA

polymerase

http://www.youtube.com/watch?v=2KoLnIwoZKU

20.2 DNA Technology – analyzing genes

Gel Electrophoresis: Use electricity to separate DNA

fragments in an agarose gel DNA is negatively charged Longer molecules travel slower than

shorter molecules

Restriction fragment analysis DNA can be digested with restriction

enzymes, and then analyzed

Genome mapping DNA sequencing – dideoxy chain termination: http://media.hhmi.org/hl/10Lect2.html?start=39:49&

end=42:08

http://www.youtube.com/watch?v=3JkL_cIRRnw Sequencing by synthesis: http://www.dnatube.com/video/2954/Pyro-Sequencing Human genome sequencing – shot gun sequencing: http://www.youtube.com/watch?v=-gVh3z6MwdU

Analyzing Gene Expression Transcription is a measure of gene expression Use probes to measure amt of mRNA present,

as a way to quantify gene expression DNA microarray assays – a grid of single

strand DNA fragments, get tested for hybridization with cDNA molecules

http://media.hhmi.org/hl/10Lect2.html?start=46:55&end=49:52

FISH

Fluorescent in situ hybridization- to determine which cells are expressing certain geneshttp://www.youtube.com/watch?v=BBQWWi6cFXU

Gene function In vitro mutagenesis Add inactive genes with a marker (mutated

genes), put the gene back into the cell so it “knocks out” the normal functioning gene

RNAi – use of synthetic double strand mRNA to breakdown mRNA or block translation; acts to knock out certain genes

Knock out mice – Mario Capecchi http://on.aol.com/video/nobel-prize-win

ning-scientist-on-knockout-mice-517890437

RNAi – use of double stranded mRNA molecules to “knock out” genes

SNP – Single nucleotide polymorphism

A single base pair site where variation is found Used as genetic markers for

particular diseases Find common genetic

marker for people who are affected with a disease

Study nearby region of DNA to look for genes involved in disease

Cloning – organisms from single cells

Plants – cells from adult plants incubated in medium can grow into normal adult plants

Animals – nuclear transplantation (i.e. Dolly) The adult cells need to be dedifferentiated Nucleus from a differentiated adult cell is

transplanted into a egg cell with the nucleus removed

Problems – defects: premature death, obesity, liver failure

Problems appear to be due to chromatin methylation issues

Stem Cells

Stem cells are unspecialized and can differentiate into specialized cells.In a stem cell, DNA is arranged loosely.

In a differentiated cell, genes not needed are shut down

Embryonic stem cells – from embryos in the blastula stage

Can reproduce indefinitely, can differentiate into many different cell types – pluripotent

Why are they valuable? have the potential to supply cells to repair

damaged or diseased organs Adult stem cells – can differentiate, but not as

widely as embryonic stem cells

Induced Pluripotency - Shinya Yamanaka took adult fibroblast cells

(connective tissue cells) Reprogrammed the cells to become pluripotent- to

being capable of differentiating into different cell types (like stem cells)

https://www.youtube.com/watch?v=i-QSurQWZo0 Reprogrammed cells with master transcription

factors

Yamanaka won the Nobel Prize in Medicine 2012

Current research with pluripotency Problems with traditional genetics

approach due to cancer causing genes Use of small compounds to mimic

transcription factors Use of drug like chemicals to enhance

reprogramming

Another way to create pluripotent cells

Haruko Obokata from Riken Center for Developmental Biology, Kobe, Japan

Stimulus-triggered acquisition of pluripotency (STAP) Took lymphocytes from mice, bathing them in acid

solution for about 30 minutes. Cultivated the cells by adding a special protein. In two to three days, the process had transformed

the cells into pluripotent cells. They developed into nerve and muscle cells.

Mouse embryo injected with pluripotent cells (labeled with fluorescent protein)

Applications of DNA Technology Diagnosis of diseases Gene Therapy Production of proteins for market Other pharmaceutical products Forensic evidence Environmental Cleanup Agricultural applications