Chapter 20: Recombinant DNA Technology: Power Tools at the Cellular Level summary

Cutting with restriction enzymes

• Restriction endonucleases (restriction enzymes) are produced by bacteria to cut DNA into small fragments.
• The enzymes help bacteria to prevent viruses.
• Every restriction enzyme cuts DNA at certain regions called restriction sites.
• If you need to transfer a human gene into a bacterial plasmid, the following steps can be followed.
  • Select a restriction enzyme that produces sticky ends after cutting DNA.
  • Slice the bacterial plasmids and human DNA using the restriction enzyme.
  • Combine the bacterial plasmids and human DNA.
  • Add DNA ligase enzyme to seal the DNA backbone.

Separation of molecules using gel electrophoresis

In gel electrophoresis, molecules are sorted based on their electrical charge and size.

DNA molecules are often separated using gel electrophoresis.

DNA is pulled through the gel because they have a negative charge.

The DNA molecules are then attracted to the positive side under the effect of an electric current.

Generation of cDNA by reverse transcriptase

• Bacteria cannot utilize genes from eukaryotes in translation without removing the introns.
• Hence, this led to the production of intron free genes called complementary DNA(cDNA).
• The steps for cDNA production are as follows:
  • Isolation of mRNA for the protein of interest.
  • Use reverse transcriptase enzyme to generate single-stranded DNA which is complementary to the mRNA.
  • Use DNA polymerase or reverse transcriptase to make a new strand form another strand for the DNA hence forming complementary DNA.

Cloning of genes into a library

• A DNA library is a recombinant DNA molecule that contains a gene of interest.
• A vector is used to clone a gene into a library.
• The process of putting a gene into a vector and cloning the gene into a library includes the following steps:
  • Cut the DNA containing the gene of interest and the vector using the same restriction endonuclease.
  • Add DNA ligase to the mixture of DNA to be cloned and the vector.
  • Put the vector in a cell population.

Types of DNA libraries

• DNA libraries
• cDNA library
• genomic library

Finding genes with DNA probes

DNA probes can be used to get vectors with specific genes of interest after cloning genes in a library.

Probes are DNA single strands that locate certain DNA sequences.

Probes are marked with a radioactive marker or a fluorescent dye to locate them in a sample of DNA.

Using PCR to copy a gene

• PCR allows the copying of the gene of interest through PCR.
• Primers are DNA sequences that are single-stranded.
• DNA polymerase utilizes the primers to start DNA replication.
• This copies the DNA located close to the gene of interest.

Using DNA sequencing to read genes

The sequencing of DNA determines the nucleotide order in the DNA backbone.

DNA sequencing uses dideoxyribonucleotide triphosphate {ddNTP}.

DNA polymerase also plays a role in copying DNA during sequencing.

Sequencing uses primers that identify the gene of interest to be sequenced.

Role of molecular biology in solving problems

• Genetically modified organisms are used in agriculture because they contain genes of various organisms. Wild plants may interbreed with modified plants transferring genes for herbicide susceptibility to weeds.
• Genetic examination of fetuses allows early detection of genetic diseases.
• Genetic examination in adults allows the identification of inherited diseases in their families.
• Human hormones like human growth hormone and insulin can be manufactured using recombinant technology using bacteria.

Identification of genes for diseases

• Genetic screening can help to identify if someone has recessive alleles for a certain genetic disease.
• The following molecular techniques can be used to check for the presence of the disease alleles:
  • Restriction enzymes can show the presence or absence of disease and normal alleles in a genotype.
  • Probes for alleles causing disease and normal alleles can show the presence of these alleles in DNA.

Fixing a gene using gene therapy

• The introduction of a gene to treat genetic diseases is called gene therapy.
• There are many barriers to human gene therapy that should be prevented:
  • Discovery of safe vectors that can be used to transfer genes into humans.
  • Procedures for transferring therapeutic genes into target populations must be developed.
  • Stem cells that generate cell populations should be identified.

Revision

Gene Cloning
Any process by which many copies of a gene are made

In Vitro
A process performed outside of a living system

In Vivo
any process performed inside of a living system

Polymerase Chain Reaction
PCR; technique for amplifying select segments of DNA by dramatically increasing the number of its copies. IN VITRO

Kary Mullis
the brain behind PCR in 1985. Winner of ’93 Nobel Prize

Denaturation
Step 1 of PCR: Mixture of nucleotides, primers, and TAQ POLYMERASE heated to break H bonds, creating single-stranded DNA

Annealing
Step 2 of PCR: Double stranded DNA (Single strands+primers) created by slight cooling. Primers attach to 3′ ends!

Primer Elongation
Step 3 (final step) of PCR: DNA polymerase extends primers and creates complimentary strands. Gene is now effectively copied.

TAQ Polymerase
Enzyme essential to PCR, extracted from Thermus aquaticus (heat-resistant) bacteria

Vector
Insertion element essential for IN VIVO placement of genetic material. Can be a VIRUS or PLASMID

Plasmid
Vector element- a small, circular piece of bacterial DNA that replicates independently

Viruses
Vector elements that enter host and inject genetic material into cells

Recombinant DNA molecules
Molecules composed of DNA from more than one source; vectors inherently contain this once manipulated

Restriction enzymes
Enzymes that “cut out” intended fragments of DNA (RESTRICTION FRAGMENTS) to be used in creating RECOMBINANT DNA

Restriction Fragments
Fragments of DNA “cut out” of an original DNA molecule by RESTRICTION ENZYMES that is to be used in creating RECOMBINANT DNA

Sticky Ends
Single stranded overhangs on a DNA molecule that H-bond with complimentary strands to form RECOMBINANT DNA

Gel Electrophoresis
Electrical process that arranges DNA fragments according to size within an agarose gel plate

Dideoxy-ribonucleotides
Nucleotides that lack an oxygen molecule and terminate replication. Used in Gel Electrophoresis in a mixture of DNA, DNA Pol., primers, and regular deoxyribonucleotides

Site-directed mutagenesis
Process that involves the introduction of particular mutations to particular DNA stretches. Vectors and blots (to confirm effects) are essential

Southern
Blot that is used to detect DNA sequences. Following electrophoresis, bands are blotted onto nylon membrane

Northern
Blot that detects RNA and indicates if a particular gene has been transcribed in a given cell

Western
Blot that detects proteins. Proteins are denatured, then marked with a corresponding antibody probe and blotted onto a nylon membrane