DNA TECHNOLOGY AND GENOMICSCHAPTER 20

P. 384-410

BIOTECHNOLOGYManipulation of organisms (or components) to make useful products

• Recombinant DNA: inserting genes into different organisms

• Fermentation: using yeast & bacteria to make cheese, wine, bread, etc

• Selective breeding: of livestock, pets, etc• Cloning: of genes, proteins, organisms• RFLP analysis: compares DNA sequences of

different people &/or species• Microassay: compares how genes are expressed in

different tissues/under different conditions

DNA CLONING: AN OVERVIEW

DNA is very large molecule; only small portion are genes

Gene Cloning: separates gene-size pieces of DNA & makes copies

• 1) bacterial plasmid isolated• 2) foreign gene inserted• 3) recombinant plasmid replicates• 4) copies of gene/protein used for practical

purposes

GENE CLONING

RESTRICTION ENZYMESCut DNA at specific sites (“restriction sites”)

• Creates several restriction fragments with “sticky ends” (single-stranded ends)

• Bonds other restriction fragments cut with same enzyme

• Bond is sealed w/ DNA ligase

DNA VECTORS

Cloning vector: original plasmid (bacterial DNA) into which foreign (human) DNA is inserted

• Used to copy desired DNA

GENE CLONING PROCEDURE1) Isolate bacterial vector & DNA fragment containing gene

2) Cut both w/ same restriction enzyme• Creates several fragments of DNA, incl. one w/

gene of interest• Vector & DNA will have complementary sticky ends

(due to S-P backbone)

3) Insert DNA into vector• Sticky ends bond together w/ DNA ligase

GENE CLONING PROCEDURE (CON’T)

4) Cloning of cells (genes)

• Use transformation to introduce cloning vector into a bacterial cell

• Use antibiotics to ensure only recombinant plasmids cloned

5) Identify clones w/ gene of interest

• Nucleic Acid Hybridization: base-pairing gene w/ another nucleic acid

• Nucleic Acid Probe: complement of gene of interest; labeled w/ radioactive isotope to easily identify

CLONING & EXPRESSING EUKARYOTIC GENES

While efficient, bacterial cloning of eukaryotic genes can be problematic:

-Gene expression simpler in bacteria

-Human DNA has non-coding regions (bacteria do not have spliceosomes)

-Bacteria can not modify proteins after translation

Can use yeast instead

-Eukaryotic, single-celled, rapid reproduction

-YAC: contains origin, centromere, telomeres; can undergo mitosis

POLYMERASE CHAIN REACTION (PCR)

Quickly amplifies (copies) DNA w/out cells (in vitro)

• 1) Heat separates DNA strands• 2) Primers bond to each single

strand• 3) Heat-stable DNA polymerase

build compliments• 4) repeat cycle

Used to increase amount of DNA in ancient organisms, crime scene samples, fetal genetic testing, etc

RESTRICTION FRAGMENT ANALYSISUses gel electrophoresis to separate DNA fragments by size

• Yields bands of DNA in specific pattern• 1) Digest DNA w/ restriction enzyme• 2) Load DNA samples into gel & run (towards + pole)• 3) Each fragment produces different band patterns

• Different alleles (sickle-cell); Compare individuals

SOUTHERN BLOTTINGReveals DNA sequences & restriction fragments

• Transfers electrophoresis bands to paper to be probed• Isolates gene of interest

• Used to compare coding sections of DNA from several organisms

• Can detect heterozygous carriers of genetic diseases

RFLP’s: Different non-coding segments of DNA

• Used to compare different organisms (unique to individuals)

• May serve as genetic markers for linkage (location on chromosome)

• Detected by Southern Blotting

HUMAN GENOME PROJECTTo determine complete nucleotide sequence of all human chromosomes (April, 2003)

3 Stages:

• 1) Genetic Mapping• Map 1,000’s genetic markers spaced evenly in each

chromosome

• 2) Physical mapping• Cuts DNA into known restriction fragments & arranges them

in order• Fragments prepared w/ cloning vectors (i.e.: YAC, BAC)

• 3) DNA Sequencing• Determine nucleotide sequence in each fragment at a time• Uses labeling, synthesis, & electrophoresis• “Sanger Method” synthesizes DNA complement to one being

sequenced (see Fig. 20.12)

GENOMICS (STUDY OF GENES)1) Identify protein-coding genes in a DNA sequence

• Most DNA is non-coding (98.5% in humans!)• Size of genome ≠ greater complexity (see Table 20.1)• Human gene expression highly regulated

2) Determine function of genes

• Disable gene & observe consequence • In vitro mutagenesis: mutate gene & observe phenotype

3) Study “Grouped” genes

• DNA Microarray Assay: shows which genes are expressed, when, and what factors may influence expression

4) Compare genomes of different species

• High degree of similarity within non-coding sequences• BLAST: database of genomes• NCBI BLAST Site

PRACTICAL APPLICATIONS OF DNA TECHNOLOGY

1) Medical

• Diagnose disease; Human gene therapy

2) Pharmaceutical

• Hormone production (insulin, human growth hormone)

3) Forensic

• DNA Fingerprinting

4) Environmental

• Bacteria & microbes clean up waste

5) Agricultural

• Transgenic animals & GMOs