CHAPTER 20 DNA TECHNOLOGY AND GENOMICS

Overview of How Bacterial Plasmids Are Used to Clone Genes

Using a restriction enzyme and DNA ligase to make recombinant DNA restriction fragments with sticky ends

Cloning a Human Gene in a bacterial plasmid

Cloning a Human Gene in a bacterial plasmid

Cloning a Human Gene in a bacterial plasmid cloning vectors-plasmids, viruses, 10,000,000 1,000 w/ DNA—10,000 w/o 100 w/ DNA—1,000 w/o 1,000,000 w/o plasmid

Cloning a Human Gene in a bacterial plasmid

Using a nucleic acid probe to identify a cloned gene denaturation nucleic acid hybridization nucleic acid probe

Expression vectors-has a prokaryotic promoter or the gene is inserted into a plasmid that has a restriction site downstream of a promoter Problem!! Introns Solution? mRNA – reverse transcriptase -- cDNA

Making a complementary DNA (cDNA) for a eukaryotic gene 3. How do you isolate? many different mRNA’s in a cell pick the right cell make all of them

Yeast artificial chromosomes-have an origin of replication, a centromere and two telomeres with foreign DNA so they behave normally during mitosis and replicate when yeast cells divide can hold much more DNA than a plasmid electroporation- first used on animal cells now on bacteria too; electricity causes a temporary pore in the cell membrane

Genomic Libraries Can also make cDNA libraries starting with all mRNA being produced by a specific cell. Advantage is that it only gives you the DNA that is coding protein in that cell. Could then make microarrays for all human genes and determine proteomics for a cell.

Polymerase Chain Reaction - PCR DNA polymerase for PCR was taken from bacteria that live in hot water; the primers are the key to which DNA gets replicated.

Gel Electrophoresis

Using restriction fragment patterns to distinguish DNA from different alleles; takes patience or luck

Restriction fragment analysis by Southern Blotting alkaline solution draws through the gel removing and denaturing some of the DNA Single stranded DNA is attached to the paper.

Chromosome Walking produces a map of overlapping restriction fragments YAC’s can carry inserted fragments that are 1,000,000 base pairs long BAC’s can carry up to 500,000 base pairs

Sequencing of DNA by the Sanger Method Step 1 Make labeled cDNA strands with special nucleotides that stop the chain when they are added

Sequencing of DNA by the Sanger Method Step 2 Different length strands are produced randomly with the ddNucleotides stopping the strand polymerization when they are added

Sequencing of DNA by the Sanger Method Step 3 The new DNA strands are separated by gel electrophoresis.

Sequencing of DNA by the Sanger Method Step 4: Read the sequence of the strands from the bands on the autoradiograph

G A C T G A A G C

Alternative strategies for sequencing an entire genome. Celera used the maps and sequence data from the public consortium

Completed in 2003, the Human Genome Project (HGP) was a 13- year project coordinated by the U.S. Department of Energy and the National Institutes of Health. During the early years of the HGP, the Wellcome Trust (U.K.) became a major partner; additional contributions came from Japan, France, Germany, China, and others. Project goals were togoals 1. identify all the approximately 20,000-25,000 genes in human DNA, 2. determine the sequences of the 3 billion chemical base pairs that make up human DNA, 3. store this information in databases, 4. improve tools for data analysis, 5. transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project.

DNA microarray for gene expression Proteomics-study of the full sets of proteins encoded by genomes Challenges: More proteins than genes Proteins differ with cell type and state Proteins are extremely variable in structure and function

DNA microarray for gene expression 2,400 human genes shows which genes are being made into protein in this cell

in vitro mutagenesis-take out the gene mutate it and put it back into the cell to see what it affects RNA interference-uses synthetic double stranded RNA with the same sequence as the mRNA that one wants to destroy; will stop viral replication in cell cultures but not in organisms Long double-stranded RNAs (dsRNAs; typically >200 nt) can be used to silence the expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, and plants). Upon introduction, the long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway. 1. First, the dsRNAs get processed into nucleotide (nt) small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer (initiation step). 2. Then, the siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process.

3. The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). 4. Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. In mammalian cells, introduction of long dsRNA (>30 nt) initiates a potent antiviral response, exemplified by nonspecific inhibition of protein synthesis and RNA degradation. The mammalian antiviral response can be bypassed, however, by the introduction or expression of siRNAs.

bioinformatics-using computers and mathematics to deal with the tremendous amount of data single nucleotide polymorphisms-single base pair variations

RFLP markers close to a gene

A possible gene therapy procedure Problems: In a multicellular organisms, it is difficult to get the gene into and expressed by enough cells to make a difference. We could eventually correct the defect in germ or embryonic cells but should we?

DNA fingerprints from a murder case RFLP markers from satellite DNA with “simple tandem repeats”

Pharmaceutical Products human insulin human growth factor plasminogen activator (clot busters) artificial vaccines Currently only made by bacteria and viruses

Hello Dolly “Pharm” animals

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

Genetically modified Golden Rice with beta- carotene Ordinary Rice

Banding patterns

Analyzing DNA

Injecting DNA into a cell