DNA Technology

Genetic Engineering

Genetic Engineering

• The direct manipulation of genes on DNA

• Has led to new biotechnology in which organisms or parts are manipulated to make useful products

• Allow for the making of recombinant DNA: a DNA molecule made from two different sources

Extraction Process Steps

1. Disrupt the plasma membrane with a detergent

2. Destroy proteins and RNA

3. DNA-containing liquid is separated from the remaining contents with a centrifuge.

4. DNA is precipitated with alcohol

How DNA is cut out

• DNA is cut out with restriction enzymes (restriction endonuclease)

• Enzymes cleave or cut out DNA at specific sites along the DNA strand-- restriction sites

• Causes staggered cuts which creates "sticky ends"

How to create recombinant DNA

• The complimentary sticky ends join to a fragment from another DNA that is cut with the same enzyme.

• DNA ligase helps to bond to new segments together to create a new recombinant DNA molecule

Gel Electrophoresis

• In order to make use of the fragments made by cleaving DNA, the individual DNA segments must be separated

• Gel Electrophoresis is the most common separation technique

• Takes advantage of the negative charge of DNA

• Can also separate other biomolecules

Gel Electrophoresis Steps

1. Restriction enzymes are used to cut DNA

2. The fragments are loaded into the gel

3. Electrical current is applied

4. The DNA fragments will move through the gel towards the positive end of the charge (move from negetive to postive ends)

5. Fragments move based on their size (larger ones are slower)

• The gel can be strained to visualize the fragments

Southern Blotting

• Used to find a particular sequence in a sample of DNA

• After separation, the fragments are transferred to a nylon membrane and bathed in a solution containing probes

• Short DNA fragments are used to find a DNA fragment of interest

• The probe is a complement and is tagged with radioactive or fluorescent dyes

• The probe attaches to the DNA sequence of interest and "tags" it or makes it visible

Polymerase Chain Reaction (PCR)

• Used to amplify (produce) specific regions of DNA

• Can make billions of copies of a particular DNA segment without using cells

• Often used when samples are very small

• DNA sample is incubated with a special DNA Polymerase, artificial DNA primers to flank the specific regions, and a supply of nucleotides

Polymerase Chain Reaction (PCR) steps

Polymerase Chain Reaction (PCR) steps

1. Denaturation - heated to separate strands

2. Annealing - cooled so primers may attach

3. DNA synthesis - warmed so polymerase can copy strands

   (video we watched that rapidly duplicated DNA)

Molecular Cloning

• It involves isolating specific sequences or genes and making new copies of that sequence

• Allows for the manipulation and study of specific genes and their protein products and non-coding regions

• The cloning of recombinant DNA usually requires a vector to help insert the new DNA into a bacteria cell where it can be copied (Usually a plasmid or a phage)

Plasmid (bacteria) Vectors

• Vectors are vehicles. They transmit an infectious agent from an infected animal to a human or another animal.

• Plasmid Vectors are used to clone SMALL PIECES of DNA

• Must have two components

• Origin of replication to allow replication inside of the bacteria

• Selectable marker - usually antibiotic resistance; allows presence of plasmid to be easily identified

• The plasmid is cut, new DNA is inserted, and the plasmid will be introduced into a bacteria cell by transformation (bacteria takes in foreign DNA and duplicates with it)

• (E. coli is often used for this process)

Phage (virus) Vectors

• Phage Vectors are larger and can be used to copy LARGER sequences

• Most common is phage lambda

• Virus will infect host cell and use it to reproduce; new DNA is incorporated into host genome (lysogenic)

• Phage genome is linear instead of circular

• (put DNA into a virus and let it reproduce through the lysogenic cycle)

DNA libraries

• Using the processes of molecular cloning, a collection of DNAs can be put together in a DNA library

• Often a genomic library - a representation of the entire genome

• DNA is fragmented and each fragment is inserted into vectors which are placed into host cells

• Each cell contains a single fragment in a plasmid or each phage contains a single fragment

• All cells or all phages together make up the library

cDNA

• Libraries often contain cDNA

• Complementary DNA produced from an edited RNA transcript

• Requires reverse transcriptase

• Made so that the DNA sample contains no introns

• More efficient for expressing eukaryotic DNA in a bacteria

• (reverse transcriptase converts RNA into DNA. That new DNA is called cDNA. c = complementary)

Reproductive Cloning

• Used to make clones of an entire organism

• Parthenogenesis - an embryo grows and develops without fertilization

• Involves replacing the haploid nucleus of an egg with a diploid nucleus from a donor cell of the same species

• The egg is then placed in a surrogate mother for development

• The first cloned animal was Dolly the sheep in 1996

• Many animals have since been cloned, but often exhibit abnormalities

• The age of the DNA may affect the life expectancy of the clone

• Attempts have been made to clone human embryos as a source of stem cells Face resistance due to bioethical issues

GMOs

• Genetically Modified Organisms

• Produced by introducing recombinant DNA into an organism

• If the foreign DNA introduced is from a different species, the organism is considered transgenic

• Bacteria, plants, and animals have been genetically modified since the 1970s for academic, medical, agricultural and industrial purposes

• (makes food more resistant to disease, animals bigger, etc)

Transformation

• Transformation of bacteria typically uses plasmids or viruses as vectors (plasmid or virus is inserted into bacteria and let sbacteria reproduce with it)

• Requires an origin of replication and a selectable marker (reporter genes) to determine which cells have been transformed

• Usually antibiotic resistance genes or gfp gene (green fluorescence)

• Almost any gene can be inserted into bacteria or yeast cells causing the cells to produce large amounts of protein product

• Requires expression vectors with extra sequences needed for the gene to be expressed

• Example - bacteria are engineered to produce human insulin and clotting factor

Genome Mapping

• Finding the location of genes on each chromosome

• Genetic maps list genes and locations

• Physical maps represent distance in nucleotides

• Data is entered into international databases to make information accessible

Genome Sequencing

• Determines the DNA sequences of a piece of or the entire genome

• Once sequenced, genomes can be analyzed and interpreted

• Important for genetic disease study

• Used to generate DNA microarrays

• DNA fragments are attached to a glass slide or silicon chip and are used to identify active genes and sequences

Manipulating Genes and Expression

• Synthetic DNA and RNA can be made and manipulated to create and study effects of mutations

• Reveals cause-and-effect relationships Could involve:

• Knockout experiments - inactivate genes

• Antisense RNA - complementary microRNAs made to block translation

• RNA interference (RNAi) - small, interfering RNAs are made to break down mRNA

DNA Technology

Using these and many other methods, biotechnology can be used for:

• Diagnosing disease

• Gene therapy

• Medicine/pharmaceuticals

• Forensics

• Environmental issues

• Agriculture