4.5: Application of reproduction + genetics

Human genome

Human genome

Project that began in 1990 using Sanger sequencing. Published a full human genome in 2003, which produced data still be analysed. Aimed to identify all genes and their original chromosome, determine base pair sequences, store this, improve analytical tools, transfer these technologies privately and address any issues.

Sanger

Sequencing method which breaks up DNA and adds complementary nucleotide triphosphates, but makes one into a dideoxynucleotide marker which stops the sequence. These chain of differing lengths are organised, terminal identified, and the base sequence is then viewable.

Dideoxynucleotide

Marker used in Sanger sequencing. Formed by removing both the 2 and 3 OH groups from a NTP. Marked using an antigen radioactive isotope or fluorescent marker. Used to compare complementary DNA strands.

Next generation sequencing (NGS)

Term for new DNA sequencing techniques. An example is passing DNA through micropores in protein molecules.

100k genome

Project launched in 2012, which sequenced 10,000 genomes of those with a rare disease or cancer, and their relatives using NGS techniques. Run by Genomics England, which aims to create and ethical and consensual programme, set up an NHS genomics programme and encourage discoveries by pairing DNA sequences with medical records.

Genetic engineering

The process of manipulating, altering and transferring genes from one organism or species to another. This makes a GM organism.

Recombinant

DNA from two different species combined.

Transgenic

Organisms that have DNA from another species introduced to their cells.

Donor

Term for DNA introduced to another organism, making that organism be transgenic and have recombinant DNA.

Gene probe

Used to identify the correct gene. Has a specific segment of single stranded DNA which is complementary to a specific gene section. Used to identify STRs during DNA fingerprinting.

Restriction endonuclease

Enzyme that cuts DNA at specific nucleotide sequences, either straight across a DNA double helix (blunt cut) or a staggered cut which leaves unpaired bases on both strands, leaving sticky ends. Leaves in introns, and can cut within the gene of interest. Also used to cut open the circular DNA molecule that makes up a plasmid.

Sticky ends

Unpaired bases left on both strands by a staggered cut, which readily pairs to complementary DNA sequences. For insulin genes, done by EcoR1 enzyme.

EcoR1

Restriction endonuclease produced by E.coli. The first to be isolated. From E.coli strain RY13. Catalyses formation of breaks in the DNA backbone of a specific sequence where a guanine nucleotide is next to an adenine nucleotide. A staggered cut.

Palindrome

Formed by EcoR1 cutting DNA. The four unpaired bases at the end of each strand are in reverse, forming this. It is also known as a recognition sequence.

Reverse transcriptase

An enzyme made by retroviruses. Produces DNA from an mRNA template, which is then known as copy DNA. DNA polymerase then catalyses the synthesis of a complementary DNA strand, making a double stranded molecule. Has no introns.

Copy

Type of DNA formed by reverse transcriptase synthesising a complementary DNA strand to the mRNA. Contains no introns. DNA polymerase then synthesises a second complementary strand, forming a double stranded molecule.

Detergent

Used to dissolve the phospholipids cell wall in order to break down bacteria in order to retrieve plasmids from cell debris. They are then used for vectors.

Sodium hydroxide

Used to make an alkaline environment within bacteria to denature membrane proteins. This is done in order to break down bacteria in order to retrieve plasmids from cell debris. They are then used for vectors.

Vector

Viruses or plasmids used as a vehicle for carrying foreign genetic material into a cell. Good ones are self-replicating, small, not broken down by host enzymes, do not stimulate immune responses, screenable to prove successful vectors and have markers to prove successful host cells.

Calcium chloride

Chemical used to increase chances of DNA taking up the plasmid. Calcium ions have a positive charge, which bind the plasmid’s negatively charged DNA backbone to the membrane lipopolysaccharide. Used alongside a heat shock - cells chilled at 4 degrees are briefly heated to 42.

Empty

Term for vectors that have not taken up the gene of interest, which is identified via DNA sequencing or blue-white screening once within a bacteria.

Marker

Gene which distinguishes the plasmid’s presence, indicating whether the DNA transfer was successful. Often antibiotic resistance genes are used, as these can be grown in an antibiotic and are shown to be successful via living.

Blue white

Method of screening which shows whether cells have taken up empty plasmids. Bacteria are grown on a medium containing lactose analogue (X-gal) and turn white if they contain the gene, but blue if the plasmid is empty.

Lactose analogue

AKA X-gal. Bacteria is grown on a medium containing this during blue-white screening. They turn white if they contain the gene, but blue if the plasmid is empty.

Cloning

The final step of GM bacteria. This occurs in large volumes within fermenters, and the insulin they produce is made in large quantities and purified before medical use.

S. mutans

Oral bacteria which produces lactic acid, contributing to tooth decay. Versions which do not produce this are available, which outcompete the original and reduce cavity formation.

Gene gun

Method of introducing a novel gene where small spheres are fired, normally gold or tungsten, coated with a preparation of the gene. Some penetrate the cell wall and are taken up through the cell membrane.

Electroporation

Method of introducing a novel gene where an electric field is used to increase the permeability of cell membranes, enhancing gene uptake.

Microinjection

Method of introducing a novel gene where a membrane is pierced by an ultra-fine needle and the gene is injected into the cytoplasm or nucleus. This is more developed for animal cells.

Agrobacterium tumefaciens

The name of the bacterial vector used to introduce a novel gene. This is the most common method for making transgenic plant cells.

T-DNA

Section of Agrobacterium tumefaciens plasmid which can integrate into the plant’s chromosome. This forms auxins which produce a gall, giving a plant crown gall disease.

Plant crown

Disease given by Agrobacterium tumefaciens Ti plasmid, which forms auxins which causes galls.

Roundup ready

A GM soybean which contains a herbicide resistance gene, allowing them to be treated without delaying their growth.

Bt

Tomatoes which have Bacillus thuringiensis (a bacterium) genes incorporated, as it codes for an insecticide protein. This is only produced in leaves, avoiding the dangers of human consumption, as shown by a 91 day rat trial.

B. thuringiensis

Bacterium incorporated into Bt tomatoes, conferring tomato hornworm, fruitworm and pinworm and tobacco hornwood resistance.

Flavr Savr

AKA anti sense. Tomatoes which have a second copy of the polygalacturonase enzyme introduced, which has a complementary base sequence to the original gene. mRNA produced is also complementary, cause them to pair in the cytoplasm which prevents translation.

Polygalacturonase

An enzyme which ripens tomatoes by breaking down pectin in their cell walls. This can cause them to over ripen over large distances. Flavr Savr tomatoes were grown to counteract this.

Pharming

The practise of producing pharmaceutical molecules in GM crop plants, such as antibodies, blood products, hormones, recombinant enzymes and human and animal vaccines.

Short tandem repeats

Repeated intron sequences, Up to 13 bases can repeat up to several hundred times. The number of repetitions is different in different individuals, and is tested for during DNA sequencing.

Polymerase chain reaction

Semi-conservative DNA replication which works fast and well with degraded and damaged samples. DNA is separated, cooled enough for primers to bind to single strands, and elongated by taq polymerase. Can be contaminated, only works on small fragments and has a limited amount of cycles before plateauing.

Taq polymerase

From a bacteria which lives in hot springs. Used in PCRs due to high temperature tolerance. Catalyses the synthesis of complementary DNA strands by adding complementary nucleotides and forming phosphodiester bonds. Has a high error rate, around 1/9000.

Primers

Single, short stranded pieces of DNA between 6-25 bases long. They are complementary to the start of the DNA strand and bind to it, signalling taq polymerase to begin. Join at 55 degrees.

Elongation

Phase of PCR where taq polymerase action begins at 70 degrees. For each fragment, two identical double strands are produced. DNA is then heated back to 95 degrees to restart the process.

Agarose gel

Used in gel electrophoresis. A polysaccharide extracted from seaweed with pores, through which small molecules, such as DNA, can move.

Gel electrophoresis

Used in Sanger sequencing and DNA fingerprinting. DNA is placed in wells at one end, and a voltage is applied. Phosphate in the DNA backbone is negative, so it moves towards the anode. Lighter, and therefore smaller, fragments move more. This allows the fragment length to be estimated.

Southern blotting

Process where a nylon membrane touches the gel and picks up DNA fragments. Probes are then attached to STRs, and a film sensitive to luminescent probes or X-rays is left over overnight. An autoradiograph then reveals a banding pattern, which is the fingerprint.

DNA fingerprinting

The process of identifying STR sequences in introns in order to show paternity or identity. Not always accurate and can be seen as a breach of privacy. Less invasive, and requires smaller samples. Composed of PCR and gel electroporesis.

Thermocycler

Where a PCR takes place due to the rapid temp changes, from 97.5, 55, 70 degrees.

Dystrophin

A protein which stabilises muscle contractions to prevent damage. Those with the sex-linked Duchenne muscular dystrophy have missing exons, causing the entire gene to be unreadable.

Exon skipping

Method of gene therapy when an antisense oligonucleotide is added which is complementary to a mutated sequence. It acts as a molecular patch for the missing exon, restoring the reading frame and allowing for a partially functional version of the gene to be synthesised. An example is drisaperson.

Gene therapy

An example of treating genetic disease where defective alleles are replaced by healthy ones, either using a vector or naked plasmid DNA. Can be somatic or germ-line therapy.

Somatic

Type of gene therapy where body cells are targeted in affected tissues, meaning changes are uninheritable.

Germ-line

Type of gene therapy where corrective genes are introduced to the oocyte. This means the change is inherited. Controversial due to the unknown way genes may interact with each other.

Genomics

Field which analyses the structure and functioning of genes, which can have major effects of healthcare. This includes more accurate diagnoses by identifying specific genes, better drug prescriptions by finding genes which affect drug metabolism and improving treatments.

Autologous

Cells from the same individual. Used in tissue engineering.

Allogenic

Cells from a donor of the same species. Used in tissue engineering.

Xenogenic

Cells from a donor of another species, such as pig cells used in cardiovascular transplants. Potential they can contain pathogenic viral sequences which can harm humans, but claimed to have these sequences eliminated. Used in tissue engineering.

Syngenic

Cells for tissue engineering that are from genetically identical organisms, such as MZ twins. Used in tissue engineering.

Scaffold

An artificial structure used to build tissues. Must allow cells to attach and move, deliver and retain cells, be porous for diffusion, and be biodegradable and absorbable.

Neotissue

Term for tissue formed by the seeding of cells onto a scaffold. Must remain as the scaffold breaks down, and should grow at the rate of the scaffold’s degradation.

Cell lines

Animal cells that can be grown indefinitely, forming clones. Requires optimum pH, temperature, oxygen, etc. Sometimes requires special conditions, such as chondrocytes (cartilage cells) needing low oxygen to mimic development in skeletal tissue.

Embryonic

Type of stem cells found in 3-5 day old embryos. Totipotent - can become any cell and form a whole organism. 100 in each blastocyst, making isolation in useful numbers easier. Easier to culture.

Induced pluripotent

Stem cells which are reprogrammed from adult stem cells. Can differentiate into most cells, but not enough to form a whole organism. Harder to isolate and has less developed technology on how to culture.

Stem cell niche

Where adult stem cells are found. Can replace normal cells lost via normal wear and tear, injury or disease, but limited. In some tissues, such as the heart, only divide under certain conditions.

Transformed

Term for a cell that has incorporated a plasmid containing the foreign gene.

Therapeutic

Type of cloning where only tissues and organs are cloned, the opposite of reproductive cloning which is whole organisms. The latter is illegal.

95, 55, 70

The temperatures PCR cycles through in the thermocycler. High to split the chains, then lowered for primers to attach and then heightened to the optimum temperature of taq polymerase.

DNA ligase

Used when a gene is added to a plasmid vector to make the join permanent, binding the sugar-phosphate backbones. This means it has been successfully spliced.