Polymerase Chain Reaction (PCR)

PCR: a technique that makes large number of DNA copies faster than DNA cloning process

DNA is heated. Heating denatures (separates) hydrogen bonding holding the dsDNA together and forms two ssDNA molecules
DNA is cooled and ssDNA primers are added. Two primers are added, each complementary to the 3′ end of ssDNA. (ssDNA ~ role of RNA primers)
DNA polymerase is added. A special, heat-tolerant DNA polymerase derived from bacteria adapted to living in hot springs is added.

DNA polymerase attaches to primers at each end of ssDNA and synthesizes complementary DNA strand
In the end one initial dsDNA becomes two dsDNA

Expressing Cloned Genes

Bacterial Expression Systems

For eukaryotic gene to be expressed must have its coding exons and a bacterial promoter
Expression vector: a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the eukaryotic gene can be inserted in the correct reading frame
- The bacterial host cell will recognize the promoter and proceed to express the foreign gene now linked to that promoter.
Bacteria have same mechanisms for replication & transcription so can express eukaryotic genes

Eukaryotic Expression Systems

Electroporation: Method of introducing recombinant DNA into eukaryotic cells → electrical pulse applied to cell → creates temporary holes in its plasma membranes so DNA can pass thru
Introduced DNA is incorporated into a cell’s genome by genetic recombination, then it can be stably expressed by the cell
- Evolution: Because of their common ancestry, all living organisms share the same basic mechanisms of gene expression.

Analyzing Gene Expression & DNA Sequencing

When trying to find mRNA for a specific gene can use nucleic acid probe: a short, single-stranded nucleic acid (either RNA or DNA) complementary to the mRNA of interest
DNA sequencing: genetic engineering technique that determines the order of nucleotides in DNA to analyze DNA through base-pairing rules

Problems With Gene Expression

When eukaryotic DNA are inserted into the genome of a bacterium, introns often prevent translation
To avoid this problem, reverse transcriptase (from retrovirus) obtains DNA with required genes directly from mRNA
- DNA obtained in this manner is called complementary DNA (cDNA) and lack the introns that suppress transcription

Medical Applications of Biotechnology & DNA Sequencing

Can use mentioned techniques to compare gene expression in healthy and diseased tissues → researchers are finding genes that are turned on or off in particular diseases

Diagnosis and Treatment of Diseases

Can use PCR with primers that target the genes associated with these disorders → amplified DNA product is then sequenced to reveal the presence or absence of the disease-causing mutation

Personal Genome Analysis

Personalized Medicine

In humans, genome-wide association studies identify and use single nucleotide polymorphisms (SNPs) as genetic markers for alleles that are associated with particular conditions
- Help ppl minimize risk for disease and better treatment through Genetic profiling
- Humans share 99% DNA, differences caused by variation of nucleotide bases

Human Gene Therapy and Gene Editing

Gene therapy: introduce new genes as a treatment for disease
- Goal: insert a normal allele of the mutated gene into the somatic cells of the tissue affected by the disorder → can now make missing protein
CRISPR-Cas9: edited cells from sickle-cell proteins with some success

Small Molecules and Drugs

Some drugs that combat tumor cells are small molecules that can inhibit a tyrosine kinase
But bcuz tumor cell have random mutations bcuz of high rate of division → drug resistance

Protein Synthesis

Scientists can use organisms to produce lots of protein products
- Human insulin gene cloning: insulin gene is inserted into bacteria; bacteria multiply and reproduce human insulin protein for use in treating diabetes