Restriction Nucleases Cut at specific recognition sequence Fragments with same cohesive ends can be...

Restriction Nucleases

• Cut at specific recognition sequence

• Fragments with same cohesive ends can be joined

Gel Electrophoresis

• DNA molecules separated by size• DNA detected by ethidium

bromide staining or by prior incorporation of radioisotope

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

Generating Labeled Probe

• Random primers annealed to DNA fragment

• DNA polymerase incorporates labeled nucleotides

• At higher temperatures only identical sequences hybridize

• At lower temperatures related sequences also hybridize

Stringency Of Hybridization

Southern And Northern Blotting

• Complex mixture of DNA or RNA separated by electrophoresis

• Transferred to membrane and hybridized with labeled probe

DNA Cloning In Bacteria

• Insert DNA fragment into bacterial plasmid

• Propagate recombinant plasmid in bacteria

DNA Library

• Collection of cloned DNA fragments• Usually are set of recombinant plasmids contained

in bacteria• Each bacterial colony contains one cloned fragment

Genomic DNA Library

• Contains entire genome of a particular individual

• DNA fragments generated by restriction nuclease

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

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

Polymerase Chain Reaction

• Oligonucleotides complementary to opposite ends of sequence to be amplified serve as primers for DNA polymerization

DNA Amplification Using PCR

•Repeated cycles of denaturation, annealing, and DNA synthesis

DNA Cloning By PCR

• Genomic DNA clones: PCR amplification of segment between primers

• cDNA clones: Reverse transcription of mRNA followed by PCR

PCR In Forensic Analysis

• PCR using primers that flank a VNTR

• Genetic fingerprint by analyzing several VNTRs

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

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

Gene Expression Patterns In Cancer Cells

• Characteristic expression patterns in different types of cancer cells

Dideoxy DNA Sequencing

• Each reaction includes one of four ddNTPs

• ddNTPs block chain growth

• Fragment sizes indicate positions of each nucleotide

Automated Sequencing Of DNA

• One reaction with four fluorescent labeled ddNTPs

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

Highly Parallel Sequencing

• Clonal amplification of DNA fragments on solid surface• High density array of clonal DNA clusters

Sample preparation

• nucleotide addition• imaging by fluorescence or chemiluminescence to detect

nucleotide incorporation at each DNA cluster

Sequencing by synthesis: repeated cycles

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

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

Western Blotting

• Complex mixture of proteins separated by polyacrylamide-gel electrophoresis and transferred to membrane

• Antibody used to detect specific protein

Producing Proteins

• Insert gene into expression vector adjacent to strong promoter

• Introduce into cells• Purify overexpressed

protein

• 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

Inheritance Patterns• Simple mendelian

Autosomal dominant

Autosomal recessive

X-linked recessive

• Complex genetic diseases: many common diseases, risk dependent upon multiple genes and environment

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

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

Gene Targeting In Mice

RNA Interference

• Turn off gene expression• Introduce dsRNA to

degrade specific mRNA

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

Generation of Mutant Organisms using CRISPR/Cas9