DNA Technology and Genomics. Recombinant DNA n Definition: DNA in which genes from 2 different sources are linked n Genetic engineering: direct manipulation.

DNA Technology and Genomics

Recombinant DNA

Definition: DNA in which genes from 2 different sources are linked

Genetic engineering: direct manipulation of genes for practical purposes

Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Bacterial genetics Nucleoid:

– region in bacterium densely packed with DNA (no membrane)

Plasmids: – small circles of DNA

Reproduction by binary fission (asexual)

Bacterial DNA-transfer processes

Transformation– genotype alteration by the uptake of

naked, foreign DNA from the environment (Griffith expt.)

Transduction– phages that carry bacterial genes from

1 host cell to another – generalized~ random transfer of host

cell chromosome – specialized~ incorporation of prophage

DNA into host chromosome Conjugation

– direct transfer of genetic material; cytoplasmic bridges

– pili; sexual

Bacterial Plasmids Small, circular, self-replicating DNA separate from

the bacterial chromosome F (fertility) Plasmid: codes for the production of

sex pili (F+ or F-) R (resistance) Plasmid: codes for antibiotic drug

resistance

Recombination of E. coli

Bacterial plasmids in gene cloning

Clone genes for insertion into organisms

Clone proteins for medical/ pharmaceutical purposes

DNA Cloning

Restriction enzymes (endonucleases)– in nature, these enzymes protect bacteria from

intruding DNA– they cut up the DNA (restriction)– very specific

Restriction site – recognition sequence for a particular restriction

enzyme Restriction fragments

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

Sticky end– short extensions of restriction fragments

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

fragments Cloning vector

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

Eukaryotic Gene Cloning Isolation of cloning vector

(bacterial plasmid) & gene-source DNA (gene of interest)

Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind fragmented DNA w/ DNA ligase

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

Cloning of cells (and foreign genes)

Identification of cell clones carrying the gene of interest

Genomic Libraries

Cloned genes from a genome are stored in a “genomic library”

Recombinant fragments in bacteria or phages

Complimentary DNA (cDNA) Library – mRNA extracted– Reverse

transcriptase makes a complimentary strand of gene

DNA Analysis

PCR (polymerase chain reaction)

Gel electrophoresis Restriction fragment

analysis (RFLPs) Southern blotting DNA sequencing

Practical DNA Technology Uses

Diagnosis of disease Human gene therapy Pharmaceutical products

– Vaccines– Hormones

Forensics– Crime scene analysis of DNA

Animal husbandry (transgenic organisms)– “Pharm” animals

Genetic engineering in plants– Disease/ pest resistance

Polymerase chain reaction (PCR)

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

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

Applications: fossils, forensics, prenatal diagnosis, etc.

DNA Analysis

Gel electrophoresis:– separates nucleic

acids or proteins on the basis of size or electrical charge creating DNA bands of the same length

Restriction fragment analysis Restriction fragment length

polymorphisms (RFLPs)– Differences in restriction

fragment patterns on homologous chromosomes

– Occur in noncoding DNA sequences

– Serve as inheritable genetic markers

Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence

DNA Fingerprinting

Southern Blotting

DNA Sequencing

Determination of nucleotide sequences – Dideoxy Chain-Termination

Method (Sanger Method)– Whole-genome approach

(Venter and Celera Genomics)

Genomics: the study of genomes based on DNA sequences

Human Genome Project– Begun in 1990; largely

completed by 2003

Genomics

The National Center for Biotechnology Information (NCBI) – Created a database of gene

sequences created by the Human Genome Project and other sequencing endeavors

– Genbank– BLAST software allows for

comparison of sequences

Analyzing Gene Expression Northern Blotting

– Gel electrophoresis done with labeling probes to determine function

RT-PCR– Uses reverse

transcriptase and PCR– Compares gene

expression between different samples

Studying Gene Interaction

DNA Microassay– Many DNA

fragments on a glass slide or chip

– Can be tested for interaction with other genes marked with fluorescent markers

Determining Gene Function

In vitro mugagenasis– Disable certain genes and

observe consequences– Mutations “knock out” certain

genes RNA interference (RNAi)

– RNA used to block translation of certain genes

Transposons transposable genetic

element; piece of DNA that can move from location to another in a cell’s genome– chromosome to

plasmid, plasmid to plasmid, etc.)

– “jumping genes”

Eukaryotic Genes

98.5% of all DNA does not code for proteins, rRNA, or tRNA

Most is repetitive DNA 44% is made of

transposable elements

Transposable Elements

Transposons– Move w/in a genome by

DNA intermediate Barbara McClintock

(1940’s and 50’s)– “Jumping genes”– Researched the location of

colored kernels in maize Retrotransposons

– Move by means of a RNA intermediate

Multigene Families In the human genome, ½ of coding DNA is in multigene

families– Collections of identical or very similar genes– Identical- ribosomal RNA molecules– Similar- α-globin and β-globin

Pseudogenes- nonfunctional nucleotide sequences (very similar to functional genes)

Genome Evolution

Duplications of chromosomes– Unequal crossing over

Duplication and divergence of DNA segments– Ancestral globin gene

present day α-globin and β-globin genes

Rearranging genes– Exon duplication/ exon

shuffling– Transposable elements