Ch 8 Cell Bio

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Impact of recombinant DNA on Medicine

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• Completion of the Human Genome Project
• Cloning & isolation of genes
• Discovery of new genes & their protein products
• Study of gene function
• Understanding of how gene abnormalities cause disease
• Study how gene expression is of regulated & the role of genes in embryogenesis, growth and development, aging, tumorogenesis.
• Alteration & re-engineering of genes to transfer back into an animal germ-line.
• Production vaccines and protein hormones
• Diagnostic confirmation in symptomatic patients
• Carrier testing for genetic disorders
• Predict response to treatment, evaluate treatment, and predict prognosis and outcome.
• Population screening to predict future genetic disease or assess the risk for complex conditions such as cancer, cardiovascular diseases & neurodegenerative disorders
• Genetic profiling of disease cells provides specific targets for development of medications, antibodies, and design of gene therapy approaches.

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Techniques used in analysis of DNA

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1. restriction enzyme digestion of DNA
2. isolation, cloning and propagation of DNA fingerprints
3. labeling and mobilization of immobilized DNA and RNA
4. nucleotide sequencing of fragments of DNA
5. genetic engineering or manipulation of DNA sequences and reintroduction into viable organisms

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restriction endonucleases

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came from bacteria (means of protecting there own genome by cleaving foreign DNA), used to cleave the phosphodiester backbone at specific sequences

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palindromic sequence

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the recognition site of the restriction endonuclease, is read the same from front and back, important because they leave overhangs (5’ overhang, 3’ overhang and blunt) (sticky ends)

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what is a DNA recombinant molecule?

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a DNA sequence that has arisen from the splicing together two or more fragments

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uses for restriction endonucleases

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can be used to map DNA or type a pattern of DNA fingerprinting

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gel electrophoresis

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used to analyze the fragments produced by restriction endonucleases, DNA has a net negative charge (which is not that different between different sized fragments), use electrophoresis to separate due to size, can use agarose (>500bp) or polacrylamide gel (small pieces)

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EtBr stain and 32P

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used to stain the fragments after electrophoresis

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cloning vectors

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DNA molecules used to transport sequences between the biological host and the test tube

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plasmid vectors

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make cloning possible, double stranded circular chromosome, found in bacteria (E. coli), self replicating and a small size

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features found on plasmid vectors

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1. origin of replication
2. antibiotic resistance gene-selectable marker
3. multiple cloning sequences containing restriction sites to facilitate cloning of inserted DNA
4. Lac-Z operon for blue/white selection, allows for selection of recombinants
5. small size for efficient transformation

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step for cloning DNA fragments

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1. insertion of a DNA fragment using restriction enzymes and ligase
2. introduce recombinant DNA plasmid into E. coli through transformation
3. allow plasmid and E. coli to replicate (can make >100 copies of plasmid in one E. coli, but one E. coli can only take up one initially)
4. screen colonies to detect presence of plasmid (antibiotic selection (kanamyacin or ampicillin), blue/white screening (disruption of LacZ operon) or size determination)
5. purification of plasmid

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DNA libraries

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constitutes an entire collection of clones, two types:
1. genomic DNA library-involves the cleavage and cloning of genomic DNA
2. cDNA library-RNA -> DNA through a cDNA (complementary DNA) step

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reverse transcriptase

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can make a DNA from an RNA

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Nucleic acid hybridization

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the process where complementary ssDNA reforms into double helices, can occur between DNA-DNA, RNA-RNA or DNA-RNA (only if complementary)

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probes

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ssDNA used to determine presence of complementary sequencesuses for hybridization reactions

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uses for hybridization reactions

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very specific so can be used to detect 1 molecule per cell if need to, can be used to detect how many copies of DNA are found in cell, also very selective and specific, can detect differences in a single base pair

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changing the stringency of the hybridization reaction

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allows for altering the specificity of the reaction, allows DNA that are not perfectly matched to hybridize

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Southern analysis

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analyze genomic DNA (gene size, genetic map, exon/intron sequence)

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Northern analysis

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examine RNA (expression of a specific gene, transcript size, abundance (absolute or relative)

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in situ analysis

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cellular expression, chromosomal location, location relative to other genes, distribution of a particular DNA or RNA molecule, within an intact cell

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microarray analysis

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novel technique. Can compare 1000’s of genes at one time. Useful for comparing gene expression between different tissue types (brain vs heart) or normal vs malignant

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applications of the different types of hybridization procedures

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- used to detect a gene of interest in a sample or library.
- used to detect whether a cell is expressing a particular RNA transcript.
- used to determine transcript size
- used to detect the abundance of a transcript within a particular tissue.
- used to map the position of introns and exons as well as the start and stop sites for RNA transcription.
- used for screening for genetic diseases (eg. RFLP genetic analysis)
- used to detect single nucleotide differences in humans, such as SNP’s (single nucleotide polymorphisms)
- used to find genes that are related but not identical by allowing a less stringent hybridization. Especially important in relating animal genes to human.

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Polymerase chain reaction

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enzymatic amplification of a region of DNA

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requirements of PCR

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1. template DNA
2. gene specific primers
3. supply of dNTPs (not NTPs)
4. DNA polymerase (Taq polymerase)
5. favorable reaction conditions (buffers, co-factors, temperature)

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3 steps of PCR

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1. Denaturation (94C)
2. Annealing (variable, depends on the primer)
3. Extension (72C)

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uses of PCR

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1. amplify DNA
2. DNA testing and typing (not much different but more sensitive than RFLP analysis)
3. clone and isolate DNA or cDNA
4. generate DNA or cDNA libraries

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DNA sequencing

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an enzymatic process using a DNA polymerase, has specific template, primer and 3’ OH group requirements, use ddNTP’s vs. dNTP’s because ddNTPs terminate the reaction (no free 3’OH) (dideoxy or chain termination reaction)

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automated DNA sequencing

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can label each ddNTP different colors to determine sequence, much faster

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limitations of DNA sequencing

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can only sequence short sequences (~750 bp)

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shotgun sequencing

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breaks the genome into fragments and using computer programs to search for overlap

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genome map

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has specific gene markers

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genetic mapping

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is based on the use of genetic techniques to construct maps showing the positions of genes and other sequence features on a genome. Genetic techniques include cross-breeding experiments or, in the case of humans, the examination of family histories (pedigrees)

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physical mapping

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uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the positions of sequence features, including genes

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genetic mapping is based on:

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1. the principles of inheritance
2. linkage analysis

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Mendel’s first law of genetics

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alleles segregate randomly, parent that is Aa has F1 that has an equal chance of getting A and a

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Mendel’s second law of genetics

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pairs of alleles segregate independently, inheritance of A is independent of inheritance of B

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genetic linkage

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found in genes found on the same chromosome, genes on the same chromosome likely to be inherited together, more like partial linkage

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partial linkage

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due mostly to crossing over, used to map relative positions of genes on a chromosome, the closer 2 genes are, the less often they will be segregated by crossing over

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recombination frequency

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a measure of the distance between two genes, smaller if shorter distance and therefore less likely to recombine

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linkage analysis

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used to determine the order of genes and the length between them,

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DNA markers

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mapped features that are not genes, must have at least two alleles to be useful, 3 types:
1. simple sequence length polymorphisms (SSLPs)
2. single nucleotide polymorphisms (SNPs)
3. restriction fragment length polymorphism (RFLPs)

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simple sequence length

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repeat sequences that display length variations, used DNA fingerprinting and parental diagnosis, different types:
1. minisatellites-AKA variable number tandem repeats (VNTRs),
2. microsatellites-simple tandem repeats

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single nucleotide polymorphism (SNPs)

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These are positions in a genome where some individuals have one nucleotide (e.g. a G) and others have a different nucleotide (e.g. a C). SNPs detection is based on hybridization analysis

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restriction fragment length polymorphisms

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restriction sites that exist as polymorphisms existing as two alleles, one allele displaying the correct sequence for the restriction site, and the second allele having a sequence alteration so the restriction site is no longer recognized, important for genetic testing to indicate a disease locus, visualized by Southern with a probe for the restriction site,

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types of physical mapping procedures

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1. restriction mapping
2. fluorescent in situ hybridization
3. sequence tag site mapping

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restriction mapping

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locates the positions for recognition sequences for restriction enzymes, constructed when use different restriction enzymes (BamHI and EcoRI) at the same time (double restriction), sizes determined through electrophoresis and order determined

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fluorescent in situ hybridization

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enables the position of a gene/marker on a chromosome to be directly visualized with a fluorescent probe

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sequence tag site mapping

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the positions of short unique sequences that occur only once in the genome being studied, mapped by PCR or hybridization analysis, common sources are SSLPs and ESTs (expressed sequenced tags, obtained from sequencing cDNA)

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in vitro transcription

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bacterial RNA polymerase is used to synthesize cRNA in vitro, SP6, T7 or T3 promoters required

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in vitro translation

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rabbit reticulocyte lysate used to synthesize proteins in vitro

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Xenopus expression system

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injection of cRNA into Xenopus oocytes, so that proteins can be synthesized in vivo

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Bacterial expression

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introduction & expression of cloned cDNA transcript into bacterial cells, abundance of protein, no post-translational modification.

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mammalian cell expression

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introduction and expression of cloned cDNA transcript into mammalian cells, use HeLa, HEK, 3T3 cells, basic mammalian vector features same as bacterial except that the promoter is optimal for mammalian cells (e.g. CMV or SV40 promoter) and the antibiotic resistance gene is different (neomycin, zeocin), post-translational can also occur

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how to study the function of proteins

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mutations
1. site directed mutagenesis
2. antisense technology and strategies
3. genetic engineering
4. conditional knockout transgenic mice

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site directed mutagenesis

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altering the nucleotide sequence of cDNA, allow single base pair mismatch, used for studying consensus sites and protein recognition sequences

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antisense technology

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used for inhibition of gene expression, antisense nucleotide sequence base pairs with a complementary sense mRNA to prevent it from translated, thought to either (a) accelerate degradation of RNA, (b) prevent introns from being spliced out, (c) preventing the exportation of mRNA, or (d) preventing ribosome binding

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uses of antisense technology

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prevent production of specific disease-causing proteins, deactivation of oncogenes, suppression of viral RNA expression (AIDS)

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dominant negative mutations

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constitutive integration of antisense genes into chromosomes to drive the constant expression of the antisense and knocking out a particular gene

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genetic engineering

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the manipulation of DNA sequences and re-introduction into viable organisms - gene insertion, replacement or deletion

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transgenic animal

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carries a foreign gene deliberately inserted into its genome, can be made to knock out or knock in a particular protein

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how to make transgenic animals

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1. targeting a gene in embryonic stem cells (ES cells) growing in tissue culture
2. injecting the desired gene into the pro-nucleus of a fertilized mouse egg

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embryonic stem cell method

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ES cells are harvested from the inner cell mass (ICM) of mouse blastocyst. ES cells can be grown in culture and are termed totipotent since they retain their full potential to produce all the cells of the mature animal, including its gametes

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outline of steps to producing a transgenic animal

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1. make recombinant DNA containing vector DNA and gene to eliminate
2. introduce this recombinant DNA into an ES cell where homologous combination occurs,
3. select for successful homologous recombination in ES cells (these are shown by resistance to neomycin
4. these successful ES cells are injected into the ICM of a mouse blastocyst
5. embryo transfer into a pseudopregnant female (may have different coat color, combo of mother and ES cell mother)
6. test the pup for the presence of the transgene through PCR
7. establish a transgenic strain

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Cre-lox strategy

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used to overcome lethality associated with knock out animals, ES cells are harvested from the inner cell mass (ICM) of mouse blastocyst. ES cells can be grown in culture and are termed totipotent since they retain their full potential to produce all the cells of the mature animal, including its gametes

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spatial conditional mice

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tissue specific Cre recombinase expression- gene gets knocked out in only one type of cell because Cre enzyme expression is controlled by tissue specific promoters. Global Cre recombinase expression in all tissues (controlled by a house-keeping” gene promoter)

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temporal conditional mice

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Inducible Cre recombinase expression controlled with tetracycline operon (on/off switch)

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the Pronucleus method

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Harvest freshly fertilized eggs before the sperm head has become a pronucleus. Inject the male pronucleus with your DNA. When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo. Implant the embryos in a pseudo-pregnant foster mother as above. The gene can also be linked to a reporter gene such as galactosidase (LacZ) or GFP, to examine the localization of the transgene in the mice