Chapter 20 DNA Technology and Genomics

Manipulating Genomes

• Genetic engineering- direct manipulation of genes for practical purposes

Ex: hormones, blood clotting factors, wine and cheese making, selective breeding of livestock,

- recombinant DNA – DNA with nucleotide sequences from 2 different sources combined in vitro

DNA Sequencing• Ultimate goal of mapping is

determining nucleotide sequences for each chromosome

• Steps of approach:1. Cut DNA from many copies of chromosome into overlapping

fragments2. Clone fragments in

plasmid or phage vectors3. Sequence fragments4. Order sequence into overall sequence

Gene Cloning

• Gene cloning – methods for preparing gene-sized pieces of DNA in multiple copies- has allowed scientists to work directly with specific genes- 2 basic purposes1. make many copies of a specific gene2. produce a protein productEx: pest resistance in plants & medical uses in large quantity (GH)

Overview of Gene Cloning• Plasmid – small ring

of DNA that carries accessory genes separate from bacterial chromosome

• Cloning vectors – original plasmid that can carry foreign DNA into a cell and replicate there

Producing Clones of CellsSteps to Cloning with a bacterial plasmid:1. Isolate the bacterial plasmid and DNA with the gene of interest.2. Both the plasmid and the DNA are digested by the same

restriction enzyme. The plasmid is cut at one specific site, while the DNA is cut many times creating fragments.

3. Allow mixing of DNA fragments with plasmids and matching of complementary sticky ends. DNA ligase is added to seal the strands together.

4. The new DNA is mixed with mutated bacteria5. Cloning begins by plating out bacteria on nutrient agar with the

target substance. - colonies will be identified by ability to reproduce in the agar

& by distinct color

Restriction Endonucleases• Restriction enzymes- cut

DNA molecules at a limited number of specific locations

-normally protect cell from intruding DNA from other organisms

-enzymes are very specific (restriction site)

- sets of restriction fragments are determined by specific enzyme

Gene Identification

• Nucleic acid hybridization – DNA of a gene can be detected by its ability to base-pair with a complementary sequence of another nucleic acid molecule

• Genomic Library – complete set of plasmid clones each carrying copies of a particular segment from initial genome

Gel Electrophoresis

• Technique using a gel as a molecular sieve to separate nucleic acids or proteins on basis of size, electrical charge, and physical properties.

-nucleic acids have negative charge from phosphate group and will travel to

positive end-sorts DNA fragments produced by

restriction enzyme digestion

Polymerase Chain Reaction (PCR)

• PCR – quicker more selective method in which any target segment of DNA can be quickly amplified.

*billions of copies can be made in a few hours*high speed and high accuracy*small amounts needed to start process*primers are very specific (Taq

polymerase)

PCR• Sources possible for

DNA amplification:a. 40,000 year old woolly mammothb. Tiny amounts of blood, tissue, and semen from crime scenesc. single embryonic cellsd. DNA in viral genes

Eukaryotic Cloning• Yeast is an ideal cloning eukaryotic genes

- easy to grow as bacteria- contains plasmids ( rare in eukaryotes)- solves incompatibility with prokaryotes

• Yeast Artificial Chromosomes (YACs) – combine essentials of eukaryotic chromosome such as origin of DNA replication, centromere, and 2 telomeres with foreign DNA

- advantage comes from the longer DNA segment carried that is more likely to contain entire gene rather than a portion

Southern Blotting

• Technique combines gel electrophoresis and nucleic acid hybridization

*reveals whether a particular sequence is present in a sample and also the size of restriction fragments that contain the

sequence* can also identify heterozygote carriers of mutant alleles associated with genetic

disorders

Restriction Fragment Analysis

• Restriction Fragment analysis – detects certain differences in nucleotide sequences of DNA molecules

Questions answered by this technique:1. Does a particular gene vary from person to

person?2. Are certain alleles associated with hereditary

disorders?3. Where in the body and when is it expressed?4. Where is gene located within genome?

RFLPs

• Restriction fragment length polymorphisms – caused by differences in restriction fragment patterns from different restriction sites on homologous chromosomes

-serves as a genetic marker for a particular location in the genome

-increased the number of markers available for mapping the human genome

Genomics

• Study of whole sets of genomes and their interactions

*more direct study of genes without inferring genotype from phenotype*Genome size generally increases from prokaryotes to eukaryotes, but does not correlate in biological complexity

Ex: 25,000 genes in human vs. 50,000 to 100,000 found in fruit fly or worm

Gene Functions

• In vitro mutagenesis- introduces a mutation in to the sequence and disables the gene or may destroy the function of the product. -Observations may reveal true function of normal gene

• RNA interference – utilizes double stranded RNA molecules that will match a particular gene sequence and trigger the breakdown or block translation

Gene Functions• Proteomics- study of full protein sets encoded by

genomes-number of proteins carrying out activities of the cell outnumbers the number of genes-Knowing when and where is vital

• Single nucleotide polymorphisms- single base pair variations in genomeoccur about 1 in 1000 base pairs-several million sites might be possible which accounts for a .1% difference between DNA sequences of like humans

20.3 Cloning and Stem Cells

Organismal cloning – cloning whole multi-cellular organisms from a single cell

- genetically identical to parent -generates stem cells

Stem cell – unspecialized cell that can reproduce itself and differentiate into one or more specialized cells

- potential for regenerating damaged tissues

Cloning Plants

• First successfully accomplished in the 1950’s with carrot plants

• Results showed that differentiation does involve irreversible changes in DNA

totipotent – ability of mature cells to “dedifferentiate” and give rise to all specialized cell types

Ex: orchids cloned for commercial purposes- pathogen resistance also possible

Cloning Animals• Differentiated cells in animals do not divide in culture –

different approach to tell if animal cells were totipotent• Nuclear transplantation – removal of nucleus of an

unfertilized or fertilized egg and replace it with a nucleus of a differentiated cell

- In studies with tadpoles it was found that the potential of transplanted nucleus to direct normal development was inversely related to the age of the donor

- older the donor the lower % of normally developing tadpoles

Ex: Dolly the sheep – identical to donor age 6, suffered from arthritis and lung disease seen in older sheep

Faulty Gene Regulation

• In most nuclear transplantation studies only a small % of cloned embryos develop normally

Reasons for low efficiency and high defects:1. Gene regulation as a result of epigenetic changes in

chromatin2. Misplaced or extra methyl groups that contribute

methylation pattern of expressed or repressed genes

3. Reprogramming of donor nuclei and restructuring of chromatin to match newly fertilized egg

Stem Cells

• Main reasons for cloning embryos is not for reproduction, but for production of stem cells to treat human diseases

20.4 Gene Therapy

• Results have been inconclusive in some cases and led to secondary issues in other cases

• Ethical questions have risen about tampering with human genes

*Eugenics – deliberate effort to control genetic makeup of humans.

• Others see no difference between transplanting somatic cells and transplanting organs

• Other questions center around interfering with evolution or genetic variation.

Medical Applications

• DNA technology has increased the identification of genes whose mutants lead to genetic diseases. -Detection may lead to ways of diagnosing, treating, and possible preventing these diseases.

Ex: HIV RNA being detected by RT PCR,Sickle cell, cystic fibrosis, Huntington’s

disease, hemophilia, and muscular dystrophy

Gene Therapy• Gene therapy is the alteration of an afflicted

individual’s genes-targets disorders that have been traceable

to a single defective gene-its believed a normal allele could be

inserted into a somatic cell of a defective gene-somatic cell must be a cell that divides

throughout patient’s life ( ex: bone marrow)

Pharmaceuticals

• Examples of DNA engineered products:1. Human insulin – 2 million people in US with

diabetes might be dependent on this drug2. Human growth hormone – targets children with dwarfism from inadequate amounts of

natural HGH3. Tissue Plasminogen Activator (TPA)- if given shortly after heart attack will dissolve blood

clots and maybe reduce risk of subsequent heart attacks

Forensic Evidence

• DNA testing in murder cases can clearly identify the suspect better than blood or tissue samples– RFLP analysis by Southern blotting can serve to

identify similarities and differences in DNA from small samples

– Provides DNA fingerprint- specific band of patterns unique to every individual

Agricultural Applications

• Transgenic animals- have gene inserted from another animal into their genome

-usually in vitro injection after fertilization, then embryo injected into surrogate mother

- Examples of goals:1. Create sheep with better quality wool2. Pigs with leaner meat3. Shorter time to maturity in cattle4. Larger muscled cattle for more meat production

Agricultural Applications• Ti plasmid- vector method

used to introduce new genes into crop plants

*Purpose could be to incorporate pest resistance, herbicide resistance, delayed ripening, or even increased nutritional value

Safety and Ethical Questions

• Concerns about DNA technology releasing or creating new hazardous pathogens led to a set of guidelines that were adopted as formal governmental regulations

• Concerns today about hazards center around Genetically Modified organisms (GMs).

Ex: salmon cloned and harvested for growth hormoneTransgenic crops

Safety and Ethical Questions

• Who should have the right to examine someone else’s genes?

• How should the information be used?• Should a person’s genome be a factor in

suitability for a job or eligibility for insurance?

• When has it been taken to far?

Mapped Genomes

• Human Genome Project – 1990-2003 completed when the nucleotide sequence of majority of DNA in each human chromosome was obtained.-researchers have also mapped sequences for E coli, yeast, nematodes, fruit flies and the mouse-Progressed in more detail in 3 stages:1. Genetic mapping2. Physical mapping3. DNA sequencing

Genetic Mapping• linkage maps – several

thousand genetic markers spaced throughout each chromosome

-order and distances between markers is based on recombination frequencies

-can be genes, RFLPs, or simple sequence DNA

Physical Mapping• Maps the distances between

markers with some physical measure, usually number of base pairs along the DNA

- cut DNA of each chromosome into restrictions fragments

-determine original order of fragments

-find overlaps between fragment ends using

probes