DNA TECHNOLOGY

Chapter 20

BIOTECHNOLOGY

•The manipulation of organisms or their components to perform tasks

•Genetic Engineering – manipulation of genes

•Gene cloning – making identical copies of a specific gene (a specific nucleotide sequence)

Techniques we will use in our labs• Transformation – putting a gene into bacteria

(Luc gene into E. coli to make them glow)• Isolation of DNA – from cheek cells• PCR – used to amplify a section of DNA (taster

gene now and later mtDNA)• Restriction Enzyme Digest – use of enzymes

to cut DNA (plasmid mapping and taster gene) • Gel electrophoresis – used to separate

different sizes of DNA fragments (plasmid mapping, taster gene, and later mtDNA)

• Sequencing – determine exact base sequence of a section of DNA (later with mtDNA)

Figure 20.1 An overview of how bacterial plasmids are used to clone genes

OVERVIEW OF GENE CLONING

• Isolation of plasmid DNA and gene to be cloned

• Restriction enzyme digest• Gene inserted into plasmid• Plasmid put into bacteria• Cells cloned with gene as part of plasmid• Identification of desired clone• Various applications

– Copies of protein product isolated– Copies of gene transferred to another

cell

Cutting DNA• In nature, bacteria cut intruding (foreign) DNA

with restriction enzymes• Enzymes cut at specific nucleotide sequences.• Recognize their own DNA by methyl (-CH3)

groups added to adenines and cytosines • Restriction Site – specific sequence that is

recognized by a specific restriction enzyme• Cuts the phosphodiester bonds (breaks

backbone)• Most sites are symmetrical: GAATTC CTTAAG

Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

• Most restriction enzymes cut in a staggered manner

G AATTC CTTAA G• The ends are called “sticky” because they

are complementary and would stick together

• Additional DNA with same sticky ends (cut with same restriction enzyme) can be inserted.

• Ligase added to make the needed phosphodiester bonds

GAATTC………..GAATTC CTTAAG………..CTTAAG

•Cloning vector – original plasmid used to carry foreign DNA into a cell and replicate there

•Bacteria most commonly used host cells

Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

•Identification of cells with clones of the right DNA gene done by using radioactive nucleic probes.

•Probes are complements to part of the gene’s sequence

•DNA must be denatured (H-bonds broken) for probe to have access to unwound DNA

Figure 20.4 Using a nucleic acid probe to identify a cloned gene

SOME PROBLEMS AND SOLUTIONS

• Problem: expressing eukaryotic genes in prokaryotic cells–Solution: expression vector – cloning vector that contains the prokaryotic promoter just upstream of restriction site and inserted gene

•Problem: Introns and noncoding regions in eukaryotes–Solution: use RNA and reverse transcriptase to make cDNA (complementary DNA or cDNA which has no introns)

•Problem: differences between eukaryotes and prokaryotes–Solution: use yeast and some fungi

Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene

•Problem: Getting DNA into eukaryotic cells–Solutions: Electroporation – shock to make holes in cell membrane and microscopically thin needles

Figure 20.x2 Injecting DNA

PCR• Polymerase chain reaction –

technique used to quickly copy (amplify) DNA and without cells–Used when DNA source is scarce

or impure–Use heat resistant DNA

polymerase, specific primers, and nucleotides to make many copies

–Used to amplify a smaller section of DNA

Figure 20.7 The polymerase chain reaction (PCR)

GEL ELECTROPHORESIS

• Gel electrophoresis – used to separate macromolecules based upon their size, charge, and other physical properties– DNA sample is cut with restriction

enzymes– Different individuals produce

different sized fragments which move through gel at different rates

– Different alleles for same gene produce different sized fragments

GEL ELECTROPHORESIS

• Restriction fragment length polymorphisms (RFLP) – differences in DNA sequences on homologous chromosomes that result in different restriction fragment patterns

• RFLP’s used as genetic markers for making linkage maps (distance between genes)

Figure 20.8 Gel electrophoresis of macromolecules

Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

SOUTHERN BLOTTING• Southern Blotting – hybridization

technique used to determine presence of specific sequences within DNA– After gel electrophoresis is done, gel

is blotted with nitrocellulose paper– DNA is transferred to paper in the

banding pattern– Paper blot is exposed to radioactively

labeled probes and then rinsed– Photographic film is laid over paper

and radioactive probes that are attached to complements expose film

Figure 20.10 Restriction fragment analysis by Southern blotting

Figure 20.17 DNA fingerprints from a murder case

Figure 20.x1a Laboratory worker reviewing DNA band pattern

Figure 20.x1b DNA study in CDC laboratory

DNA SEQUENCING

•DNA sequencing – uses dideoxy chain termination– Each copied strand starts with same

primer and ends with a dideoxyribonucleotide (ddNTP)

– ddNTPs terminate strand because they do not have 3’ OH

– Each ddNTP is fluorescently labeled differently (A, T, C, and G)

– Labeled strands are sorted by size in a gel– Light source reads fluorescence, which

determines base (A, T, C, or G)

DNA sequencing

DNA Microarrays

•DNA microarrays – numerous amounts of small DNA strands of known sequences fixed to a glass slide or chip

• Unknown DNA is labeled with fluorescent tags

• Labeled DNA is washed over array and complementary strands bond and glow

DNA Microarrays

Cloning• Cloning plants: single-cell cultures

– Works because plant cells can de-differentiate

• Cloning animals: Nuclear transplantation– First successful cloned mammal, Dolly, in

1997– Procedure does not have high success

rate– Often clones have problems– Probably due to gene regulation

differences in adult and embryo DNA

•Stem cells – relatively unspecialized cells that can both reproduce itself independently and, under appropriate conditions, differentiate into specialized cells–Found in embryos, skin, hair, eyes, dental pulp

• Potentially used to treat many diseases like Alzheimer’s, Parkinson’s, Huntington’s etc.

APPLICATIONS OF BIOTECHNOLOGY• Gene therapy – replacing defective genes with

a normal gene– In bone marrow cells, embryonic cells, and

gametes– Problems:

•Even if gene expressed, activity diminishes•Appropriate amounts expressed/ right

time/ right place•Ex. SCID (severe combined immuno

deficiency) lacks protein– Attempts to insert gene into bone

marrow cells of 10 children worked in 9 patients, but caused leukemia in 2 (inserted into cell division gene)

Figure 20.16 One type of gene therapy procedure

• Pharmaceutical products – Insulin– Human growth hormone– Tissue plasminogen activator (TPA)

•Helps dissolve clots after heart attack/stroke

– Make genetically engineered proteins to block or mimic cell receptors•Experimental drug that mimics receptor protein that HIV binds to so it attaches to drug instead of entering T-cells

• Forensic Science – DNA fingerprinting• Environmental – using bacteria to clean up

toxic wastes (extract heavy metals)• Agricultural – vaccines, antibodies, and growth

hormones– Bovine growth hormone to make produce

more milk– Genetically modified organisms (GMO)

• Transgenic organisms – organisms that are given gene(s) from another species

• Plants –genes for resistance to herbicides, less spoilage, larger fruits etc.

• Medicine – using SNPs (single nucleotide polymorphisms) as markers; helps to identify disease causing alleles

Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants

Figure 20.20 “Golden” rice contrasted with ordinary rice

Genes from daffodils and a bacterium that is inserted into rice plants so that rice can make beta-carotene

• Sheep with gene for human blood protein

• Protein is in sheep milk and isolated

• Helps inhibit enzyme that destroys lungs in CF patients

Figure 20.18 “Pharm” animals