Chapter 19 - ChromatinChapter 19 - Chromatin

•DNA PackagingDNA Packaging•histone protein histone protein

•NucleosomeNucleosome •””beads on a string” basic beads on a string” basic unit of DNA packingunit of DNA packing

•HeterochromatinHeterochromatin•highly condensed highly condensed interphase DNA interphase DNA

•(can not be transcribed)(can not be transcribed)•EuchromatinEuchromatin

•less compacted interphase less compacted interphase DNA DNA (can be transcribed)(can be transcribed)

Chromatin Chromatin - - complex of DNA and proteinscomplex of DNA and proteins

Chapter 20Chapter 20

DNA Technology DNA Technology & & GenomicsGenomics

Recombinant DNARecombinant DNA Def:Def: DNA in which DNA in which

genes from 2 different genes from 2 different sources are linkedsources are linked

Genetic engineering:Genetic engineering: direct manipulation of direct manipulation of genes for practical genes for practical purposespurposes

Biotechnology:Biotechnology: manipulation of DNA manipulation of DNA or organisms to or organisms to perform practical perform practical tasks or provide tasks or provide useful productsuseful products

Bacterial plasmids in gene cloningBacterial plasmids in gene cloning

DNA CloningDNA Cloning Restriction enzymes (endonucleasesRestriction enzymes (endonucleases):):

– in nature, these enzymes protect bacteria from in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA intruding DNA; they cut up the DNA (DNA (DNA scissors)scissors) very specificvery specific

Restriction siteRestriction site::– recognition sequence for a particular recognition sequence for a particular

restriction enzymerestriction enzyme Restriction fragmentsRestriction fragments::

– segments of DNA cut by restriction enzymes in segments of DNA cut by restriction enzymes in a reproducable waya reproducable way

Sticky endSticky end::– short extensions of restriction fragmentsshort extensions of restriction fragments

DNA ligaseDNA ligase: : – enzyme that can join the sticky ends of DNA enzyme that can join the sticky ends of DNA

fragmentsfragments Cloning vectorCloning vector: :

– DNA molecule that can carry foreign DNA into DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial a cell and replicate there (usually bacterial plasmids)plasmids)

Restriction Enzymes Restriction Enzymes (Endonucleases)(Endonucleases)

Steps for eukaryotic gene cloningSteps for eukaryotic gene cloning

Isolation of (bacterial plasmid / Isolation of (bacterial plasmid / cloning vectorcloning vector) & gene-source ) & gene-source DNA (gene of interest)DNA (gene of interest)

Insertion of DNA into the cloning Insertion of DNA into the cloning vector using the same vector using the same restriction enzyme; bind the restriction enzyme; bind the fragmented DNA with DNA fragmented DNA with DNA ligaseligase

Introduction of cloning vector Introduction of cloning vector into cells into cells (transformation by (transformation by bacterial cells)bacterial cells)

Cloning of cells Cloning of cells (and foreign (and foreign genes)genes)

Identification of cell clonesIdentification of cell clones

CloningCloning

DNA Analysis & GenomicsDNA Analysis & Genomics

PCR PCR (polymerase chain (polymerase chain reaction)reaction)

Gel Gel electrophoresiselectrophoresis Restriction fragment Restriction fragment

analysis (RFLPs)analysis (RFLPs) Southern blottingSouthern blotting DNA sequencingDNA sequencing Human genomeHuman genome projectproject

Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)

Amplification of any Amplification of any piece of DNA without piece of DNA without cells (in vitro)cells (in vitro)

Materials: heat, DNA Materials: heat, DNA polymerase, polymerase, nucleotides, single-nucleotides, single-stranded DNA primersstranded DNA primers

Applications: fossils, Applications: fossils, forensics, prenatal forensics, prenatal diagnosis, etc.diagnosis, etc.

DNA AnalysisDNA Analysis

Gel electrophoresisGel electrophoresis: separates nucleic acids or proteins on the : separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the basis of size or electrical charge creating DNA bands of the same lengthsame length

Restriction fragment analysisRestriction fragment analysis

Restriction fragment length polymorphisms (RFLPs)Restriction fragment length polymorphisms (RFLPs) Southern blottingSouthern blotting: process that reveals sequences and : process that reveals sequences and

the RFLPs in a DNA sequencethe RFLPs in a DNA sequence DNA FingerprintingDNA Fingerprinting

DNA SequencingDNA Sequencing

Determination of Determination of nucleotide sequences nucleotide sequences (Sanger method, (Sanger method, sequencing machine)sequencing machine)

Genomics: the study of Genomics: the study of genomes based ongenomes based onDNA sequencesDNA sequences

Human Genome Human Genome ProjectProject

Practical DNA Technology UsesPractical DNA Technology Uses

Diagnosis of diseaseDiagnosis of disease Human gene therapyHuman gene therapy Pharmaceutical products Pharmaceutical products

(vaccines)(vaccines) ForensicsForensics Animal husbandry Animal husbandry

(transgenic organisms)(transgenic organisms) Genetic engineering in plantsGenetic engineering in plants Ethical concerns?Ethical concerns?

FISH TechniqueFISH TechniqueFluorescence In Situ Hybridization

O.J. Simpson murder case - 1995 Odds of blood in Ford Bronco not being R. Goldman’s:

6.5 billion to 1 Odds of blood on socks in bedroom not being N. Brown-Simpson’s:

8.5 billion to 1 Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:

21.5 billion to 1 Number of people on planet earth:

7+ billion Odds of being struck by lightning in the U.S.:

2.8 million to 1 Odds of winning the Ohio lottery:

76 million to 1 Odds of getting killed driving to the gas station to buy a lottery ticket

4.5 million to 1 Odds of seeing 3 albino deer at the same time:

85 million to 1 Odds of having quintuplets:

85 million to 1 Odds of being struck by a meteorite:

10 trillion to 1