Recombinant DNA and Genetic Engineering

Chapter 12

Lipoproteins

• Form when certain blood proteins combine with cholesterol

• High-density lipoproteins (HDLs)

• Low-density lipoproteins (LDLs)

Familial Hypercholesterolemia

• Gene encodes protein that serves as cell’s LDL receptor

• Two normal alleles for the gene keep blood level of LDLs low

• Two mutated alleles lead to abnormally high cholesterol levels & heart disease

Example of Gene Therapy

• Woman with familial hypercholesterolemia

• Part of her liver was removed

• Virus used to insert normal gene for LDL receptor into cultured liver cells

• Modified liver cells placed back in patient

Results of Gene Therapy

• Modified cells alive in woman’s liver

• Blood levels of LDLs down 20 percent

• No evidence of atherosclerosis

• Cholesterol levels remain high

• Remains to be seen whether procedure will prolong her life

Genetic Changes

• Humans have been changing the genetics of other species for thousands of years

– Artificial selection of plants and animals

• Natural processes also at work

How Does DNA Recombine in Nature?

• Sexual Reproduction Recombines DNA

– Crossing over, independent assortment, fertilization

• Transformation May Combine DNA from Different Bacterial Species

• Viruses May Transfer DNA between Species

Discovery of Restriction Enzymes

• Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack

• Discovered bacteria have an enzyme that chops up viral DNA

Specificity of Cuts

• Restriction enzymes cut DNA at a specific sequence - pallindromes.

“a man a plan a canal panama”

“madam im adam”

• Number of cuts made in DNA will depend on number of times the “target” sequence occurs

Making Recombinant DNA

Using Plasmids

• Plasmid is small circle of bacterial DNA

• Foreign DNA can be inserted into plasmid

– Forms recombinant plasmids

– Plasmid is a cloning vector

– Can be used to deliver DNA into another cell

Using Plasmids

How Is Biotechnology Used in Forensics?

• The Polymerase Chain Reaction Amplifies DNA

– PCR copies a specific DNA sequence

Polymerase Chain Reaction

• Sequence to be copied is heated

• Primers are added and bind to ends of single strands

• DNA polymerase uses free nucleotides to create complementary strands

• Doubles number of copies of DNA

Polymerase Chain Reaction

PCR primers

• Short sequences that DNA polymerase recognizes as start tags

• To carry out PCR, must first determine nucleotide sequences just before and after the gene to be copied

• Complementary primers are then created

PCR DNA Polymerase

• Most DNA polymerase is denatured at high temperature

• Polymerase used in PCR is from bacteria that live in hot springs

Temperature Cycles

• DNA is heated to unwind strands

• Cooled to allow base-pairing with primers and complementary strand synthesis

• DNA is heated again to unwind strands

• Cycle is repeated over and over again

Tandem Repeats

• Short regions of DNA that differ substantially among people

• Many sites in genome where tandem repeats occur

• Each person carries a unique combination of repeat numbers

• SO: differences in short DNA segments can identify individuals “DNA FINGERPRINTING”

Short Tandem Repeats

How Is Biotechnology Used in Forensics?

• Gel Electrophoresis Separates DNA Segments

• DNA Probes Are Used to Label Specific Nucleotide Sequences

• A DNA Fingerprint Is Unique to Each Person

RFLPs

• Restriction fragment length polymorphisms

• DNA from areas with tandem repeats is cut with restriction enzymes

• Because of the variation in the amount of repeated DNA, the restriction fragments vary in size

• Variation is detected by gel electrophoresis

Gel Electrophoresis

• DNA is placed at one end of a gel

• A current is applied to the gel

• DNA molecules are negatively charged and move toward positive end of gel

• Smaller molecules move faster than larger ones

Analyzing DNA Fingerprints

• DNA is stained or made visible by use of a radioactive probe

• Pattern of bands is used to:

– Identify or rule out criminal suspects

– Determine paternity

DNA Fingerprints

• Unique array of DNA fragments

• Inherited from parents in Mendelian fashion

• Even full siblings can be distinguished from one another by this technique

12.4 How Is Biotechnology Used in Agriculture?

• Many Crops Are Genetically Modified

– The Desired Gene Is Cloned

– Restriction Enzymes Cut DNA at Specific Nucleotide Sequences

– Cutting Two Pieces of DNA with the Same Restriction Enzyme Allows the Pieces to Be Joined Together

• Using plasmids to insert DNA into a plant cell

12.6 How Is Biotechnology Used for Medical Diagnosis and Treatment?

• DNA Technology Can Be Used to Diagnose Inherited Disorders

– Restriction Enzymes Cut Different Alleles at Different Locations –sickle cell

– Different Alleles Bind to Different DNA Probes-cystic fibrosis

• DNA Technology Can Be Used to Treat Disease

12.6 How Is Biotechnology Used for Medical Diagnosis and Treatment?

– Using Biotechnology to Treat Cystic Fibrosis –deliver a working copy of the chloride pump via cold viruses

– Using Biotechnology to Cure Severe Combined Immune Deficiency – deliver working copy of receptor to stem cells of bone marrow via virus. But- leukemia.

12.7 What Are the Major Ethical Issues of Biotechnology?

• Should Genetically Modified Organisms Be Permitted in Agriculture?

Are Foods from GMOs Dangerous to Eat?

– Not, but allergens? Pesticide use?

Are GMOs Hazardous to the Environment?

– Escape of genes into wild relatives-corn, fish

Dolly and Clone

Eugenic Engineering

• Selecting “desirable” human traits

• Who decides what is desirable?

• 40 percent of Americans say gene therapy to make a child smarter or better looking would be OK

The Human Genome Initiative

Goal - Map the entire human genome

• Initially thought by many to be a waste of resources

• Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes

• Sequencing was completed ahead of schedule in early 2001

Making cDNA

Using Human Genes

• Even with gene in hand it is difficult to manipulate it to advantage

• Viruses usually used to insert genes into cultured human cells but procedure has problems

• Very difficult to get modified genes to work where they should

Basic Research

Recombinant DNA technology allows researchers to:

– Investigate basic genetic processes

– Reconstruct life’s evolutionary history

– Devise counterattacks against rapidly mutating pathogens

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