BIOL 1107 - DNA Tools and Biotechnology

DNA technology

main technologies for sequencing and manipulating DNA

Genetic engineering

direct manipulation of genes for practical purpose

Gene’s sequences

Gene’s sequences can be determined by DNA sequencing

What can be obtained by DNA cloning

-Multiple copes of DNA segments can be obtained by DNA cloning

• plasmids can be modified to become recombinant DNA molecules, and the inserted into bacteria

• such a plasmid is called a cloning vector

• it can be used to amplify a gene sequence, or to produce a protein

DNA can be cut by…

bacterial restriction enzymes. they usually produce fragments with stick ends (sequence of single-stranded DNA) that facilitates recombination

Gel electrophoresis

-the study of fragments can be done by gel electrophoresis

• DNA fragments are added close to the negative electrode and move towards the positive one. Small fragments move faster than large fragments

Polymerase Chain Reaction (PCR)

-DNA sequences of interest can be amplified by Polymerase Chain Reaction (PCR)

• probed for the target sequence are used. Only fragments corresponding to the probes will be replicated and amplified

Issues with cloned genes

-cloned genes from eukaryotes can be expressed in bacterial cells, but some issues must be overcome

• differences in promoters or regulation sequences. this is why the cloned sequences contain an expression vector (highly active bacterial promoter)

• presence of introns in many eukaryotic sequences. Researchers often used cDNA (complementary of mRNA, thus, without introns)

• otherwise, eukaryotic organisms can be used instead (ex: yeast)

Use of DNA technology to study gene expression and function

-analysis of when and where a gene or group of genes is expressed can provide important clues about gene function

-this can be done by:

• looking for mRNA expression

• using reverse transcriptase-PCR

• we can study expression of interacting groups of genes by RNA sequencing

• other methods (like microarray assays) are still used nowadays to compare expression patterns at different times, at different places and under different conditions

Looking for mRNA expression

using complementary molecules, it is possible to detect a specific mRNA (with a specific sequence)

Using reverse transcriptase-PCR

-mRNA is reversed and transcribed into cDNA

-then, a PCR is run to amplify the gene of interest (if it is expressed)

-it only shows the presence or absence of the mRNA in the sample

We can study expression of interacting groups of genes by RNA sequencing

-RNAs are isolated, cut, converted to cDNA and sequenced

-the method does not rely on known genomic sequences, and it can measure levels of expression of genes

Disabling sequences

-it is possible to determine function of genes by disabling sequences

• in vitro mutagenesis is the introduction of a mutation into a cloned gene, which alters or destroys its function

• when put back to a cell, the study of phenotype tells the function of the gene

CRISPR-Cas9

new techniques such as CRISPR-Cas9 are powerful because it is possible to target specific sequences to “knock out” genes or to repair genes

Single nucleotide polymorphisms

-some sequences such as single nucleotide polymorphisms (SNPs) are useful genetic markers since they are often associated to particular genetic disorders

• they can be targeted by tests

Clones

genetically identical to the “parent” that donated a single cell

Stem cells

relatively unspecialized cells that can reproduce indefinitely and can differentiate into one or more types of specialized cells

Totipotent

In plants, mature cells can dedifferentiate, which allows cloning. The cell becomes totipotent (ex: it can generate a whole organism)

For animal cells

-for animal cells, it mostly depends on the age of the nucleus used

• an embryonic nucleus can usually generate a whole organism

• it is much more difficult with a fully differentiated cell. Many clones will not develop or may die prematurely

• even clones do not necessarily look exactly the same

Stem cells are…

-stem cells are relatively unspecialized

• embryonic stem cells (ES) can differentiate into a variety of specialized cells. They are pluripotent

• adult stem cells can generate multiple types of cells, but not all

Induced pluripotent stem cells (iPS)

-cells that have received extra-copies of stem cell master regulatory genes.

-They undergo dedifferentiation to act (almost) like ES cells.

-They may be used for treatments against some diseases

Biotechnology

the manipulation of organisms and their components to make useful products

Medical application

-we can identify human genes related to specific genetic diseases

-we can target the products of some genes for prevention or therapy

-PCR use

-SNP use

-gene therapy

-but issues remain such as gene regulation, no interference with other cell functions,…

-and it rises ethical questions

PCR can be used to…

identify specific sequences related to genetic disorders

SNPs may be linked to…

disease-causing mutations. individuals can be tested for them

Gene therapy

the introduction of genes into an individual in order to correct the deleterious allele (ex: correct for bone marrow cell deficiency)

Pharmaceutical products

-DNA technology and genetic research help develop useful drugs

-recombinant cell cultures are commonly used to produce pharmaceutical products (ex: insulin, growth hormone)

-transgenic animals received a gene from another species. they can express this gene and act as a pharmaceutical factory

Forensic evidence and genetic profiles

-DNA testing can identify individuals with a high degree of certainty

-each individual has a unique set of genetic markers (genetic profile), especially short tandem repeats (STRs)

-the probability of two people to have the same STR markers without being identical twins is exceptionally small

-it can be used on crime scenes, to identify victims, to determine child paternity, etc.

Environmental cleanup

it is possible to modify the metabolism of microorganisms to use them to treat toxic waste material

Agricultural applications

-DNA technology is used to improve productivity (plants and animals)

-it rises concerns about ethics and potential dangers

DNA technology should be…

used with humility and caution