Recombinant DNA Technology …artificial manipulation of DNA.
–isolate DNA, –cut DNA into workable sized fragments, –amplify the fragments for storage and subsequent analysis, –identify and isolate specific sequences, –characterize by size genomic location and sequence. Recombinant DNA Technology classic
Cutting the DNA ~3,100,000,000 base pairs per human haploid genome, –average chromosome length is ~13 million base pairs, DNA is cut with bacteria-derived enzymes, The act of cutting is termed “digestion”.
Restriction Enzymes …proteins that recognize specific, short nucleotide sequences and cut DNA at those sites, …bacteria contain over 400 such enzymes and recognize and cut over 100 DNA sequences.
Palindromes stir grits no lemon, no melon 5 ’ -------G-A-A-T-T-C--------3 ’ 3 ’ -------C-T-T-A-A-G--------5 ’
Restriction Fragment Length Sizes (predicted) - IF - 25% A 25% T 25% G 25% C and Random Distribution of Nucleotides - THEN - Distance between cut sites is equal to 4 n bases, (n = number of base pairs in the recognition site) i.e. p(specific base) =.25
Average Restriction Fragment Length n = 4, 256 base pairs n = 6, 4096 base pairs n = 8, 65.5 kb base pairs
DNA is Profligate …sticky ends with complementary base pairs can form hydrogen bonds, …with DNA from any species.
Requires a Vector…...a specialized DNA sequence that can enter a living cell,...signal its presence to an investigator,... and provide a means of replication for itself and the foreign DNA it carries. Cloning …specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study.
And, Vectors… …contain unique restriction sites to facilitate the creation of recombinant DNA molecules,... must also possess a distinguishing physical characteristic such as size or shape by which it can be purified away from the host cells genome.
Vectors PlasmidE. coliup to 15 kb, PhageE. coliup to 25 kb, CosmidE. coliup to 45 kb, BACE. coli100-500 kb, YACYeast250-1000 kb.
Step 1…restriction digests and ligation of fragments into cloning vectors,
Cloning Step 3...vectors contain selectable markers, –only cells that contain vector DNA will survive selection, –recombinant vectors can be discerned from empty vectors by additional markers. Blue/White Cloning
Amplification …if you were to grind me up with a giant mortar and pestle, and extract all of my DNA coding for a single gene, you’d get about 1 g of DNA, …two liters of bacterial culture carrying my gene in a recombinant DNA vector, would yield an equivalent mass of DNA.
Assignment Read 10.1 and be prepared to clone a fragment of DNA using a plasmid as a vector.
Today More DNA Science DNA Amplification II, –Polymerase Chain Reaction, –6.7, pp. 237-239, Gel Electrophoresis, –Southern Blots, Northern Blots, –6.6, pp. 237-238, Clones and Libraries, pp
Polymerase Chain Reaction …invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino, "It was quiet and something just went, Click!" Kary B. Mullis Nobel Laureate, 1993 Chemistry
Mullis…... “PCR is a chemical procedure that will make the structures of the molecules of our genes as easy to see as billboards in the desert and as easy to manipulate as Tinkertoys”. DNA
Making DNA: Components CellPCR ss DNA template helicase, etc.? dNTPspresent Primerprimase? DNA Polymerase DNA polIII ? Environmentnucleustest tube
Oligonucleotides specific primers...short pieces of synthetic DNA can be manufactured that contain any sequence, …template specific! ~ Odds of a Specific Sequence 20-mer: 9.1 x 10 -13
Making One Strand Of DNA Add Polymerase Add dNTPs, etc. add primer
Making Two More Strands Must Denature Separate Strands
Denaturing can’t use helicase in vitro …DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases, …dNTPs are not affected by denaturation, …primers are not affected by denaturation.
Making Two More Strands Add polymerase, etc. add primer to second strand
Denaturation Step Bad …several rounds of in vitro replication could be performed (prior to PCR), however, accumulation of denatured polymerases quickly poisoned the reactions.
…bacteria discovered in a hot spring in Yellowstone Natural Park in 1965, …lives in salty water that ranges from 70 o - 75 o C, …thus, does DNA replication at very high temperatures.
Thermus aquaticus’ Enzymes …basic research demonstrated that many enzymes isolated from Thermus aquaticus function at very high temperatures, …temperatures nearing 100 o C, …DNA denaturating temperatures.
Click …Kary Mullis realized that repetitive rounds of DNA synthesis could be performed by using a heat-stable polymerase, … Thermus aquaticus: Taq polymerase.
Syllabus Update 6.1, 6.6 - 6.8, 10.1 for Friday, –read the rest of the chapter for review, –Master the Chapter 6 summary, –Master 6.8, 6.13, 6.18, 6.19, 10.6, 10.7, 10.9, 10.11. Read the Science Paper for Monday.
Genetics …in the news. …a hardcopy of the assigned paper (Monday) will earn you two of the 12.5 points on Wednesday’s quiz.
Options? This? Seymour Binzer’s brilliant phage recombination experiment that revealed gene structure? Or This? Development of the technology used to rapidly identify emerging virus? A (H1:N1 infoA (H1:N1 info)
PCR Applications …new applications are created every day, PCR products can be used for mapping genes, PCR products can be used as probes, PCR products can be probed, PCR can be used to identify genotypes, PCR can be used to sequence DNA directly.
Molecular Probing Heterologous Hybridization …genes (DNA), or gene products (RNA) can be identified based on hybridization to labeled molecules, …DNA probes are short, single-stranded stretches of nucleic acid that are complementary to target nucleic acids, –10 - 1000s of base pairs in length, …radioactive or fluorescent labeled for detection.
Probe Add a “labelled” dNTP to an in vitro synthesis reaction.
Genomic vs cDNA...Jeff’s gene, i.e. hemoglobin… isolate red blood cells, –red blood cells make lots of hemoglobin, thus the mRNA is enriched for hemoglobin sequences, construct a cDNA library, isolation of hemoglobin clones is facilitated, –genomic: ~1 of 1,000,000 clones, –cDNA from red blood cells: 1 of 3 clones.
cDNA Libraries …provide a ‘snap-shot’ of the genes expressed in a particular cell, at a particular time, or under specific condition, …however, do not provide regulatory sequences.
Cycle Sequencing Chain Termination Template ddNTPsdNTPs Lots of each sized fragment are produced, each with a specific florescent base on the end.base etc. Another Template
Dosage Compensation …more Chapter 7 X chromosomes in females provide twice the genes, as in males, –Drosophila: female genes are expressed at 50% of the male levels, –Mammals: one X ho,olog in females is silenced.
Canadian Cat Scientists Sees it First Barr Body
Lyon Hypothesis Mary Lyon; in humans, X chromosomes from father and mother are randomly inactivated.
X Inactivation The structure of the chromosome is altered. Barr Body
X-Linked Mosaicism Different cell lineages contribute to different body locations on the body.
Epigenesis A change in gene regulation brought about without a change in DNA sequence, –often to the structure of the chromosome, –or through modification of the nucleotide bases, –or through post transcriptional regulation.