chapter 20 dna technology and genomics. overview of how bacterial plasmids are used to clone genes
TRANSCRIPT
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
denaturationnucleic acid hybridizationnucleic 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 complementaryDNA (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 allmRNA being producedby 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 - PCRDNA 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 attachedto the paper.
Chromosome Walking
produces a map of overlapping restriction fragments
YAC’s can carry inserted fragments that are 1,000,000
base pairs longBAC’s can carry up
to 500,000 base pairs
Sequencing of DNA by the Sanger MethodStep 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 to 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 genomesChallenges:More proteins than genesProteins differ with cell type and stateProteins 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 organismsLong 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 20-25 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 caseRFLP markers from satellite DNA with
“simple tandem repeats”
Pharmaceutical Productshuman insulinhuman growth factorplasminogen activator (clot busters)artificial vaccines
Currently only made by bacteria and viruses
Hello Dolly
“Pharm” animals
Using the Ti plasmid as a vector forgenetic engineering in plants
Genetically modifiedGolden Rice with beta-carotene
Ordinary Rice
Banding patterns
Analyzing DNA
Injecting DNA into a cell