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+ DNA Technology and Genetic Engineering Human protein production RFLP analysis Human genome project Transgenic organisms Forensic analysis

DNA Technology and Genetic Engineering

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Human protein production. Transgenic organisms. Forensic analysis. Human genome project. RFLP analysis. DNA Technology and Genetic Engineering. DNA Technology and Genetic Engineering (making changes in DNA). Nucleus. Chromosome. 1: Production of human proteins 2: To identify people - PowerPoint PPT Presentation

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DNA Technology and Genetic Engineering

Human proteinproduction

RFLP analysisHuman genome

project

Transgenicorganisms

Forensic analysis

+ DNA Technology and Genetic Engineering (making changes in DNA)1: Production of human proteins

2: To identify people

3: To identify human diseases

4: To identify all human genes

5: To genetically engineer foodSupercoils

Coils

Nucleosomes

Histones

Nucleus Chromosome

DNA

Cell

+ Use 1: To produce human proteins in bacteria (E. coli)Example: Curing Pituitary Dwarfism

+What is Dwarfism?

Dwarfism is a recessive disease that causes adults to be no more than 4 feet tall

Little people produce little or no growth hormone, which is made by the pituitary

Researchers studied families with dwarfism and found that people with dwarfism have defective copies of the gene, GH1

+Early attempts to treat Dwarfism

Attempts to inject growth hormone from pigs (a strategy that worked previously for insulin) did not work – only GH from humans would work (until 1982 source from human cadavers and up to 20,000 pituitaries were needed!)

Some of these pituitaries were contaminated with prions, which cause degenerative brain disorders

+Solution: Use recombinant DNA to produce GH in bacteria!

Recombinant DNA = DNA that results from combining DNA from different sources ex. mouse + human DNA human + bacterial DNA

+Plasmidisolated

1Bacterium

Bacterialchromosome

Plasmid

2DNAisolated

Cell containing geneof interest

DNAGene ofinterest

3 Gene inserted into plasmid

Recombinant DNA(plasmid)

4 Plasmid put intobacterial cell

Recombinantbacterium

5

Copies of gene Copies of protein

Clones of cellGene for pestresistanceinserted intoplants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Protein used to make snow format highertemperature

Cell multiplies withgene of interest

Recombinant DNA Overview

+How Do You Make Recombinant DNA?

How do you make recombinant DNA? We need A) To isolate genes with restriction enzymes B) A vector C) To combine the genes and the vector

What do we do with it? D) Transfer the recombinant DNA to the host E) Find the gene of interest (human growth

hormone)

+ A) Isolate genes with restriction enzymes (DNA scissors)

Occur naturally in bacteria – why? Bacteriophages

infect bacteria Cut up foreign DNA

Hundreds are purified and available commercially

Recognize and cut at specific base sequences in DNA (usually 4-8 bases long)

+ Products generated by restriction enzymes

A) Sticky-end cutters

Enzyme Recognition site DNA after cuts

B) Blunt-end cutters

Enzyme Recognition site DNA after cuts

5’...G3’...CTTAA

AATTC...3’ G...5’

5’...CCC3’...GGG

GGG...3’CCC...5’

5’...CCCGGG...3’3’...GGGCCC...5’

SmaI

EcoRI5’...GAATTC...3’3’...CTTAAG...5’

+ A) Isolate genes with restriction enzymes (DNA scissors)

Take genomic DNA (in this case, human cells) and cut it with a particular restriction enzyme

MANY restriction fragments formed

ALL parts of the DNA are cut whenever there is a restriction site

Now what? Put these fragments in a vector

+ B) Vectors

Vector = something to carry the gene of interest into the host (i.e. bacteria) A. Mechanical – micropipettes or gene

guns B. Biological – virus or plasmid

Plasmid – additional, free-floating ring of DNA found only in bacteria Can replicate within a cell (has

origin of replication) Has antibiotic resistance gene

Cut both the plasmid and gene with the same restriction enzyme and their ends will hydrogen bond = gene splicing

+

C) Combine the gene of interest and the vector by sealing ends with DNA ligase… now you have made recombinant DNA

DNA1

Restriction enzymerecognition sequence

Restriction enzymecuts the DNA intofragments

Sticky end

2

3

4

5

Restriction enzymecuts the DNA intofragments

Addition of a DNAfragment fromanother source

Two (or more)fragments sticktogether bybase-pairing

DNA ligasepastes the strand

Recombinant DNA molecule

+

C) Combine the gene of interest and the vector – a differentpicture

+D) Transfer the Recombinant DNA to the Host (Transformation)Recombinant DNA is transferred to a host cell.

Can use heat-shocking or electricity to get plasmid into bacteria

How do we know which bacterial cells have our plasmid? Antibiotic resistance gene! Resistance gene allows ONLY those bacteria with the

plasmid to grow in media that have an antibiotic on it

When the host cell copies its DNA (replicates), it also makes a copy of the plasmid. Results in clones, which are genetically identical copies.

+D) Transfer the Recombinant DNA to the Host

We use bacteria because they reproduce very quickly and have all the protein synthesis machinery (enzymes, ribosomes) Insulin for Type I diabeticsBlood factor VIII-hemophilia (clotting

factor)Antigens for vaccines (Hep B, flu,

meningitis)Cutting chromosomes in order to study

individual pieces

Host Cells Produce Protein Products – ex. GH and insulin

+E) Find the gene of interest

Each clone consists of identical cells containing one fragment (of many) of human DNA

The collection of all the cloned DNA fragments is known as a genomic library Each fragment represents

a “book” that is “shelved” in plasmids inside bacterial cells

Thus, it is a library of all the organism’s genes Figure 12.6

Genome cut up with restriction enzyme

Recombinantplasmid

OR

Recombinantphage DNA

Bacterialclone

Phageclone

Phagelibrary

Plasmidlibrary

+ E) *Side note – libraries can also be made from cDNA

The enzyme reverse transcriptase can be used to make a smaller library, called a cDNA library

This contains only the genes that are expressed (transcribed) by a specific cell type (rather than ALL the genes found in an organism)

These genes can then be digested and placed in vectors

Why is this useful? Bacterial mRNA does not have introns – doesn’t have

the machinery to splice eukaryotic genes Can help a researcher study the genes responsible for

specialized functions of a certain cell type

+

Transcription1

CELL NUCLEUS

DNA ofeukaryoticgene

RNAtranscript

mRNA

Exon Intron Exon Intron Exon

TEST TUBE

Reverse transcriptase

cDNA strand

cDNA of gene(no introns)

RNA splicing(removes introns)

2

Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand

3

Breakdown of RNA4

Synthesis of secondDNA strand

5

+E) Find the gene of interest

How do we find the right “shelf” in the library?

If we know at least part of the DNA sequence, we can create a nucleic acid probe

Probe = radioactively labeled single-stranded DNA piece that can base pair with a particular sequence

Ex: ATCCGA

The probe is mixed with clones that have been heated or treated with a chemical to separate the DNA strands

The probe will “tag” the correct “shelf” or bacterial clone that contains the gene of interest

+E) Find the gene of interest

Radioactiveprobe (DNA)

Mix with single-stranded DNA fromvarious bacterial(or phage) clones

Single-strandedDNA

Base pairingindicates thegene of interest

+E) Find the gene of interest

Bacterial colonies containingcloned segments of foreign DNA

Radioactive DNA

Transfercells tofilter

1Solutioncontainingprobe

Treat cellson filter toseparateDNA strands

2 Add probeto filter

3 ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Hydrogen-bonding

Autoradiography4

Developed filmColonies of livingcells containinggene of interest

Compare autoradiographwith master plate

5

Master plate

Filterpaper

The bacterial colony can then be grown and the protein of interest can be isolated in large amounts

+E) Isolating the gene of interest

Isolate DNAfrom two sources

1E. coli

Cut both DNAs with the same

restrictionenzyme

2

Plasmid

Human cell

DNA

Gene VSticky ends

Mix the DNAs; they joinby base-pairing

3

Add DNA ligaseto bond the DNA covalently

4

Recombinant DNAplasmid Gene V

Put plasmid into bacteriumby transformation

5

Clone the bacterium6

Bacterial clone carrying manycopies of the human gene

+Mass-Produced Genes