Gn311H Final

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Type II Restriction Endonucleases

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Recognize a specific nucleotide pair sequence in DNA, then cleave DNA within that sequence
* sticky ends via staggered cuts (HindIII)
* blunt ends via straight cuts (PvuII)

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Gel Electrophoresis

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Separates DNA/RNA based on size, or proteins by size and charge

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Blotting

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Transfers nucleic acid or protein from a gel to a special membrane to be treated with a labeled probe/antibody for visualization

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Southern Blot

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Separate DNA by fragment length

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Northern Blot

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Separate RNA by fragment length

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Western Blot

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Separate protein by charge, weight, etc.

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Cloning Vectors are...

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...DNA molecules that carry DNA of interest (foreign). They can be prokaryotic or eukaryotic

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Essential features of a cloning vector

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1) unique cloning site (insertion/polylinker site)
2) stable origin of replication (Ori)
3) selectable marker (Amp^r)--help determine if plasmids successfully transformed. Antibiotic resistance can be conveyed by a plasmid so that only bacteria containing that plasmid survive on media containing antibiotics
4) reporter gene (lacZ)--induced by IPTG transcription creates B-galactosidase, which cleaves X-gal substrate to produce a blue substance (PLASMID W/ INSERT)
* no lacZ: white bacteria

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DNA library

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Overlapping set of clones spanning a region of interest

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Genomic DNA library

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contain set of DNA clones that contain whole genomes

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cDNA library

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restricted to DNA sequences that are created from mRNA using reverse transcriptase for 1st strand and DNA polymerase for double stranded

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Polymerase Chain Reaction

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Used to make many copies of a small segment of DNA
* complementary primers are needed; provide 3'-OH end for extension
* useful for applications that require large amounts of DNA or when there is only a very small amount of DNA available (fetal, trace evidence)

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PCR Steps

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1) 92-95 degrees C: denature DNA into 2 template strands
2) 55 degrees C: anneal oligonucleotide primers to template (3'-OH)
3) 70-72 degrees C: Taq polymerase extends and synthesizes new strand

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Chain-Termination DNA Sequencing

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"Sanger Sequencing"
- requires ssDNA template, a DNA primer, a DNA polymerase, radioactively/fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation
- DNA sample divided into 4 sequencing rxns, each with 4 standard dNTPs and DNA polymerase
- 1/4 ddNTPs added to each rxn (lack 3'-OH, thus terminating DNA strand extension and resulting in various DNA fragments of varying length)

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DNA Typing

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- RFLP Analysis
- Y-Chromosomal Analysis
- Mitochondrial Analysis
- STR Analysis

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STR Analysis

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- 13 core loci currently used for discrimination in CODIS
- Independently assorting loci so the product rule applies
- CODIS has local, state, and national databases
- Analysis by gel and *capillary electrophoresis (better)

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A number of diseases are associated with __________.

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Unstable trinucleotide repeats (Fragile X, Huntington's, Myotonic Dystrophy)

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When is gene therapy used?

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Only when there are no other treatments available.

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Gene Therapy

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- Introducing functional gene copies into individuals with 2 defective copies.
- Somatic cells (non-heritable) are currently used; treats but doesn't cure

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Transgene

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Introduced copy of a gene in gene therapy; if successful, will synthesize missing gene product and restore normal phenotype

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In gene therapy, _____ genes must be introduced into cells.

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Wild-type

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Retroviral vectors

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Integrate into DNA of host cell
(+) transgene transmitted to all progeny cells
(-) may integrate into gene and disrupt function

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Adenoviral vectors

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Are present only transiently in host cells and replicate autonomously (no integration)
(-) immune system can eliminate viruses along with infected cells
(+) no potentially harmful integration step

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Transgenics

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Plans or animals in which a foreign/exogenous gene has been introduced into its genome, thereby altering its genetic constitution

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Genomics

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- The study of how genomic information is utilized to produce a phenotype
- The study of whole genomes--all genes, the interactions among them and with the environment

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Genome Science

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Aims to study structure, function, and evolution of whole genomes

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Central Dogma of Genomics

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Genome --> Transcriptome --> Proteome --> Phenome

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Personalized Medicine

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- Taking into account the molecular events underlying a disease and your "genotype" to determine your best treatment strategy
- A person's genes may affect medication responses (you may or may not be getting the full benefit of a drug)
- i.e. Warfarin: individual dosage sensitivities greatly vary

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Human Genome Project: Map-Based Sequencing

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- Collins
- relies on detailed genetic and physical maps to align sequenced fragments

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Human Genome Project: Shotgun Sequencing

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- Venter
- small-insert clones prepared directly from genomic DNA and sequnced
- done by computer programs
- most of genome is sequenced 10-15 times

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Contig

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A set of overlapping fragments that form a continuous stretch of DNA

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Single Nucleotide Polymorphisms

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(SNPs) A site in the genome where individual members of a species differ in a single base pair. SNPs are valuable markers in linkage studies because they are linked to disease alleles. The greatest diversity of SNPs is in African people because it is the origin of humanity.

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Haplotype

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A specific set of SNPs and other genetic variants observed on a chromosome. New haplotypes arise from mutation or recombination.

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Linkage Disequilibrium

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- Nonrandom association of certain genetic variants (alleles) with each other, so that these combinations occur more frequently in the population than expected based on random assortment.
- If a SNP is physically close to a disease-causing locus, it will tend to be inherited with the disease-causing allele.

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Orthologs

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Homologous sequences found in different organisms. They evolved from a common ancestral gene via speciation.

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Paralogs

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Homologous sequences found in the same organism. They are produced via gene duplication within a genome.

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Microarrays

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- Nucleic acid hybridization--using a known DNA fragment as a probe to find a complementary sequence (tagged cDNA pairs with complementary probe).
- A reporter sequence encodes an easily observed product that is used to track the expression of a gene of interest.
- Each spot contains multiple identical strands of DNA, and each spot represents one gene.

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Microarrays can be used to determine...

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...the level of gene expression between tissues or different strains of an organism. These genes could be used to determine resistance, and this info could then be used for antibiotics.