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Transcript of Restriction Nucleases Cut at specific recognition sequence Fragments with same cohesive ends can be...
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Restriction Nucleases
• Cut at specific recognition sequence
• Fragments with same cohesive ends can be joined
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Gel Electrophoresis
• DNA molecules separated by size• DNA detected by ethidium
bromide staining or by prior incorporation of radioisotope
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Nucleic Acid Hybridization
• Complementary strands of DNA or RNA can renature• Labeled single stranded DNA probe often used to
detect specific DNA or RNA molecules in a sample
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Generating Labeled Probe
• Random primers annealed to DNA fragment
• DNA polymerase incorporates labeled nucleotides
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• At higher temperatures only identical sequences hybridize
• At lower temperatures related sequences also hybridize
Stringency Of Hybridization
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Southern And Northern Blotting
• Complex mixture of DNA or RNA separated by electrophoresis
• Transferred to membrane and hybridized with labeled probe
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DNA Cloning In Bacteria
• Insert DNA fragment into bacterial plasmid
• Propagate recombinant plasmid in bacteria
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DNA Library
• Collection of cloned DNA fragments• Usually are set of recombinant plasmids contained
in bacteria• Each bacterial colony contains one cloned fragment
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Genomic DNA Library
• Contains entire genome of a particular individual
• DNA fragments generated by restriction nuclease
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cDNA Library
• Contains only DNA sequences that are transcribed into mRNA
• cDNA generated from mRNA using reverse transcriptase
• Different library generated from every cell type
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cDNA Vs. Genomic DNA Clones
• cDNA clones useful for deducing protein sequences• Genomic clones useful for obtaining noncoding and
total genome sequences• Library screening to select clones of interest
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Polymerase Chain Reaction
• Oligonucleotides complementary to opposite ends of sequence to be amplified serve as primers for DNA polymerization
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DNA Amplification Using PCR
•Repeated cycles of denaturation, annealing, and DNA synthesis
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DNA Cloning By PCR
• Genomic DNA clones: PCR amplification of segment between primers
• cDNA clones: Reverse transcription of mRNA followed by PCR
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PCR In Forensic Analysis
• PCR using primers that flank a VNTR
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• Genetic fingerprint by analyzing several VNTRs
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Genetic Test Using Allele-Specific Oligonucleotides
A probe
S probe
P1 P2 C1 C2 C3 C4
A allele
S allele
-CCTGAGGAG-
-CCTGTGGAG-
-globin genePCRPrimer
• PCR, gel electrophoresis, hybridization
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Hybridization Using Microarrays
• Slide with array of DNA probes• Genomic DNA or mRNA
sample (mRNA→cDNA)• DNA sample labeled with
fluorescent dye and hybridized• Fluorescence measured
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Gene Expression Patterns In Cancer Cells
• Characteristic expression patterns in different types of cancer cells
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Dideoxy DNA Sequencing
• Each reaction includes one of four ddNTPs
• ddNTPs block chain growth
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• Fragment sizes indicate positions of each nucleotide
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Automated Sequencing Of DNA
• One reaction with four fluorescent labeled ddNTPs
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Sequencing Genomes
Shotgun method:
• Generate several genomic libraries with different size inserts
• Perform sequencing reactions on millions of genomic clones
• Establish order on chromosome based on sequence overlaps
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Highly Parallel Sequencing
• Clonal amplification of DNA fragments on solid surface• High density array of clonal DNA clusters
Sample preparation
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• nucleotide addition• imaging by fluorescence or chemiluminescence to detect
nucleotide incorporation at each DNA cluster
Sequencing by synthesis: repeated cycles
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Finding DNA Sequences That Encode Proteins
– Search open reading frames, splice sites, regulatory elements
– Compare to cDNA sequence database
– Compare to other species
• From genome:
• From cDNA: usually one open reading frame
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Antibodies to Detect Proteins
• Antibodies are produced by immune system• Billions of forms bind to different antigens• Primary antibody recognizes specific antigen;
secondary antibody used for detection
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Western Blotting
• Complex mixture of proteins separated by polyacrylamide-gel electrophoresis and transferred to membrane
• Antibody used to detect specific protein
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Producing Proteins
• Insert gene into expression vector adjacent to strong promoter
• Introduce into cells• Purify overexpressed
protein
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• Locate disease genes using physical markers• Physical markers have known locations and are polymorphic• Examine relationship between physical markers and disease • Co-inheritance indicates nearby location
Linkage Analysis
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Inheritance Patterns• Simple mendelian
Autosomal dominant
Autosomal recessive
X-linked recessive
• Complex genetic diseases: many common diseases, risk dependent upon multiple genes and environment
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Genome-Wide Association Studies
• Compare frequency of SNP alleles in healthy and disease populations
• SNP allele with higher frequency in disease population indicates genetic risk factor
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Identification of Genetic Risk Factors
• age-related macular degeneration• asthma• bipolar disorder• coronary artery disease• Crohn’s disease• diabetes-type 1• diabetes-type 2• obesity• prostate cancer• rheumatoid arthritis
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Gene Targeting In Mice
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RNA Interference
• Turn off gene expression• Introduce dsRNA to
degrade specific mRNA
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Genome Engineering Using CRISPR-Cas9
• Targeted genome editing• Introduce Cas9 (nuclease) and gRNA for genomic target• DS break, repaired by NHEJ, usually inactivates gene• Introduction of repair template allows precise editing
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Generation of Mutant Organisms using CRISPR/Cas9