Key terms in gene technology

Bacteriophage: A virus cloning vector used to insert foreign genes into bacteria. Most common ones are lambda- and M13-phages.

Bacterial artificial chromosome BAC: An artificially created bacterial plasmid, which includes all the areas needed for replication (ORI and the needed coding DNA). It copies itself only once or twice inside the host cell, but compared to a yeast artificial chromosome BAC is more stable, easy to transform, easier to clean and grows well in E. coli.

Cap-structure: mRNA is capped. The cap-structure is added to the 5'-end of the pre-mRNA during transcription, and seems to be involved in several aspects of pre-mRNA and mRNA metabolism, in most cases related to identifying the 5'-end of the RNA.

Cloning vector: A sequence to which foreign genetic material has been attached, and which then is transferred into a host cell. Most commonly the vector is a bacterial plasmid or a virus. Forget mathematical vectors or vector graphics, this vector is more of a "vessel". Plasmids can carry inserts up to 10 bp, bacterial artificial chromosomes up to 300 and yeast artificial chromosomes up to 200-2000 bp.

Codon/anticodon: A codon is a base triplet in the mRNA, and anticodon is its counterpart in tRNA. By matching an anticodon to a codon the correct amino acids can be added to the protein chain. Only the two first bases are important, causing a wobble-effect (the third codon can be different, and still the amino acid is recognized correctly). Each protein synthesis starts with a start codon (AUG) and ends in a stop codon (UAG, UGA, UAA).

Cosmid vector: A plasmid to which a cos-area from a lambda-phage (see bacteriophage) has been inserted. The cos area enables packing the cosmid inside a protein coating of the phage. Used commonly when building cDNA-libraries. 

DNA ligase: An enzyme which attaches two single strands of DNA together. It does not create a dual-stranded DNA, but only elongates an existing single strand by adding another piece to it. It is used in DNA transcription to close the gaps between Okazaki fragments, and when attaching DNA strands cut with restriction enzymes back together or to a vector. Needs ATP and Mg2+ to work.

DNA polymerase: A DNA polymerase is a cellular or viral polymerase enzyme that synthesizes DNA molecules from their nucleotide building blocks. DNA polymerases are essential for DNA replication, and usually function in pairs while copying one double-stranded DNA molecule into two. DNA polymerases also play key roles in other processes within cells, including DNA repair, genetic recombination, reverse transcription. (Wikipedia)

Expression vector: A vector built specifically for producing proteins. An expression vector includes the needed DNA-insert for coding the protein, a promoter to start the DNA transcription and start and end sites for the actual protein synthesis. Expression vectors are host cell specific.

Exon: A part of a gene which codes proteins. When a finished DNA-strand is spliced, exons are included in the end product. Some exons may be left out to achieve different variations of the gene.

Intron: A part of a gene which does not code any protein, or include functional DNA. Introns are left out when splicing.

Structure of a nucleotide.
(c) Wikipedia

Nucleotide: Consists of three parts: a base, a 5-ring sugar and 1-3 phosphates. Nucleotides with sugars with a hydroxyl group in their 3' carbon are ribonucleotides, while those with only hydrogen in 3' carbon are deoxyribonucleotides. In DNA and RNA, there are five nucleotides: adenine, uracil, thymine, cytocine and guanine. A and C are purines, G, T and U pyrimides. Purines always pair with pyrimides: A-T, C-G and A-U. Nucleotides are joined to one another with phosphodiester-bonds.

PCR (polymerase chain reaction): A method to replicate DNA automatically in a test tube using DNA polymerase. It has three stages: DNA denaturation, binding of the primers and DNA synthesis. A simple animation of it can be found from the website of Wiley. Many polymerases are sensitive to the Mg2+ concentration, so it has to be optimized for the polymerase used in the PCR.

Peptide bond: A bond formed between two amino acids in protein synthesis, when a carboxyl group and an amino group bind together and one molecule of water (H20) is lost.

Plasmid: A small, circular dual-strand of bacterial DNA, which is often used as a cloning vector. They replicate independently inside the bacterial cell. Plasmids always have a point of origin (ORI), which is the starting point for the replication. Plasmids altered for use in biotechnics have a cloning site, which includes restriction sites for several different restriction enzymes. Recombinant plasmids also have a selection gene, which is used to distinguish recombinants from wild types.

Polysome: A site for protein synthesis. Polyribosomes (or polysomes) are a cluster of ribosomes, bound to a mRNA molecule. Many ribosomes read one mRNA simultaneously, progressing along the mRNA to synthesize the same protein. (Wikipedia)

Primer: An RNA/DNA sequence serving as a start point for DNA replication. It's 18-22 bp long, and can be designed and built artificially. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand. Primers are used in PCR to give the polymerase a place to bind to. Primers are "independent" strands, which bind to the DNA to be used as a model, while promoters are sequences in the actual DNA.
Promoter: An organism-specific site where the polymerase enzyme binds and where the DNA- or RNA-replication starts. There are three types of promoters: 1. constitutive promoters, which work all the time in every tissue type in the organism, 2. tissue- or time-specific promoters which work only in some tissue types or during a certain time, 3. inducible promoters, which work only when a certain stimuli is present. (PatentLens) Eucaryotic cells have a promoter called TATA box, located 25 bases upstream from the start point of the transcription. Promoters can control the efficinecy of RNA synthesis.

RNA types: rRNA: ribosomal RNA. Exist in ribosomes, where they hold the mRNA steady and assist in protein synthesis. mRNA: messenger RNA is a copy of genetic information (DNA), which is used as a template for protein synthesis. tRNA: transfer RNA partakes in protein synthesis by matching a base triplet (a codon) to a correct amino acid, which it brings to the synthesis site.

Reading frame: Reading frame means how the DNA/RNA is "split" into three bases for reading. A sequence of AACTGTAC could be read for example as AAC|TGT|AC or as A|ACT|GTA|C. If there are over 50 amino acids between the start and stop codons, the protein has an open reading frame.

Enzymes used in recombinant DNA technology.
(c) Lecture material, origin unknown.

Recombinant DNA/RNA: Recombinant DNA/RNA includes foreign DNA/RNA, which has been inserted into the genome artificially or naturally via fagosytosis. A recombinant vector or a cloning vector is a sequence to which foreign genetic material has been attached, and which then is transferred into a host cell. Most commonly the vector is a bacterial plasmid or a virus.



Replication: A process where a dual-strand DNA is opened, and both strands are used as a template to create a new dual-strand DNA. The process is semiconservative, meaning that both new strands have 50 % of the original genetic material.

A replication fork. (c) Wikipedia

Restriction enzyme: An enzyme, which recognizes a specific sequence of DNA and cuts it. The recognized sequence is called a restriction site. Chromosomes and bacterial plasmids have hundreds or thousands of restriction sites for different enzymes. Restriction enzymes are very useful in gene technology, where the genes can be cut from precise points for sequencing or ligating to another chromosome. Restriction endonucleases cut the strand from the middle, while exonucleases cut it from the ends of the strand.

Restriction site: A palindromic site where a restriction enzyme makes the cut. Restriction sites 4 bases long appear approximately once every 300 nucleotides, so enzymes using these cut the DNA into very short strands. Restriction site of 6 bp appear once in 4000 nucleotides, and those with 8 bp very rarely.

RNA structure: One stranded, consists of adenine, uracil, cytocine and guanine. The 3' carbon of the sugar ring has a hydroxyl group (-OH). It may bind on its own, creating loops and hairpins.

Transformation of bacteria: The process of a bacteria accepting pure DNA into its cell. If the inserted DNA includes an ORI site, the DNA will be replicated inside the cell. The plasmid determines how many copies are made. Before transformation the bacteria has to be made competent, i.e. ready to accept DNA. This can be done by CaCl-treatment, which causes the cells to swell. Then pure DNA is added into the competent cells in a water solution.

Transformation efficiency: How many transformants (recombinant plasmids) are created per a microlitre of foreign DNA introduced.

Translation: The process of translating an mRNA into a protein. Watch a clear animation of it by St. Olaf's college.

UTR: Untranslated region. UTR 5' and UTR 3' are found between a gene-coding region and its promoter / poly-A -tail.

Genomic selection

Genomic selection in animal breeding means that genomic information is used together with phenotypic information to calculate estimated breeding values. Genomic information in itself is rather useless. It only shows what genetic markers, QTLs, SNPs or microsatellites one animal has. Only by connecting those markers to wanted traits can we select the animal with the "right" markers. For example, we may know that a marker X1 is connected with high milk yield, and X2 with susceptibility to metabolic illnesses. Then we can choose animals with X1 but not X2. Genetic mapping is the key to connect genetic information with phenotypic observations. However, once the traits have been linked with genetic markers, pure genotype information is enough to value an animal. In practice, genomic estimated breeding value GEBV always combines genotypic data with pedigree information.

Using genomic information increases accuracy in all traits.

Single nucleotide polymorphisms, SNPs, are used in genomic selection. At one time, current genotyping technology can identify up to 800 000 SNPs from a given DNA sample. Those SNPs are evenly distributed to the DNA strand. SNPs are known to be connected to productive traits. Animals are genotyped, their genes are valued based on the SNPs, and then best animals are selected for breeding. We don't need to know where or what the actual wanted genes are - we just know which SNPs are connected to them, and use those as a basis for selection. It's a bit like orienteering: you see from the map where you are and where you have to go, even if you have no clue in which city or country you're in!


Illumina is a manufacturer of bead chips, chips which are used to identify SNPs from a genome. For example, the  PorcineSNP60 chip has 65000 evenly spaced probes for recognizing SNPs. It can be used for four pig breeds: Duroc, Landrace, Pietran, and Large White. More information about the chip can be found from the product information sheet.

Simulations

There are two ways for evaluating genetic variation in quantitative traits: simulations and experimental genetics. Simulations are cheap and easy to repeat, but the assumptions used in the simulation may not be realistic.Experimental genetics uses real data, but is more cost and labour intensive. Simulations are carried out with different computer programs. They estimate population evolution under different conditions. Each simulation is based on real data about

• allele and genotype frequencies
• linkages between markers and genes
• population and family structure
• effective population size
• reproduction parameters
• selection (if any)
• isolated / open population
• DNA alteration parameters

Simulations work with four basic forces of evolution: mutation, recombination, selection and genetic drift. Mutation adds diversity, recombination creates new combinations of alleles and breaks linkage between chromosomal regions, selection favours some genotypes with selective advantage and genetic drift makes allele frequencies fluctuate over generations. The key for simulations is that the researcher can change the underlying variables, and study the impact of (for example) population size or family structures to genetic evolution. For a free simulation software, you can try QMsim by Sargolzaei and Schenkel (http://www.aps.uoguelph.ca/~msargol/qmsim).

4N_eμ is an important term in simulations. According to professor Alban Bouquet, in a mutation drift equilibrium the SNP (or allele) frequency depends on the variable 4N_eμ.  If 4N_eμ < 1, the distribution is U-shaped. Large % of markers are fixed, and diversity is low. When 4N_eμ  = 1, the distribution is uniform, and when it's > 1, the distribution is curved out: only a few markers are fixed, and there's high diversity. In most mammals 4N_eμ is about 0,001, so most loci are fixed at MDE and many markers need to be simulated.

Comparing the effectiveness of genomic and traditional selection

(c) informedfarmers.com

In animal breeding, there are four selection pathways: sire of bull, sire of cow, dam of cow and dam of bull. Each pathway has their own selection intensity i, accuracy r_TI and generation interval L. For bovines, dams of bulls have the highest selection intensity (around 2 %), because only the best of the best are chosen. A few more sires of bulls are chosen, about 5 %, while as much as 80 % may be chosen for dams of cows. With strict selection and reliable genomic information the genetic gain is largest when dams and sires are selected at one year of age. In such a juvenile scheme (JS), genetic gain increases while rate of inbreeding decreases. Genetic gain increases the most with strictest selection intensities, but rate of  inbreeding is also higher. (Bouquet & Juga 2012)

The study by Bouquet and Juga (2012) showed that with JS, using a multiple-ovulation embryo transfer (MOET) herd of 75 heifers genetic gain is significantly increased.  MOET does not significantly increase inbreeding rates if over 33 AI sires used. Doubling the number of flushed heifers may change response to selection with over 50 AI sires, but only if the number of genotyped females is increased as well. Increasing the number of flushings per heifer from 2 to three increases both genetic and rate of inbreeding, and may not thus be advisable. All in all, MOET and JS combined impact production traits very strongly, but functional traits only little. Compared to traditional pedigree-based selection, the variance of response is slightly increased in JS schemes whereas genetic gain istremendously increased.

(c) eadgene.info

For pigs genome information also increases the accuracy of breeding values. In a study by Tribout, Larzul and Phocas (2012) a simulation generated 39% - 58% more accurate EBVs with genomic breeding schemes. Annual genetic gain increased 63% - 128%. There was variation in the results between different traits. Rate of inbreeding was reduced 49% - 60%.  They conclude that genomic breeding schemes can increase accuracy and genetic gain while decreasing rate of inbreeding without a need to modify current breeding scheme structures. 

Last minute checks

Last quick checks before the exam on biotechnology...

Daughter design: One possible population structure when studying the QTLs of dairy cows. Genotype and markers are assessed on daughters of sires heterozygous for the markers. (Weller, Kashi, Soller 1990)

Epistasis: The function of one gene is affected by many other genes, also known as modifier genes.

Granddaughter design: One possible population structure when studying the QTLs of dairy cows.  Marker genotype is determined on sons of heterozygous sires and quantitative trait value measured on daughters of the sons. (Weller, Kashi, Soller 1990)

Interval: The chromosomal area or distance between two genetic markers. Can be measured by calculating recombination frequencies without interference in several cross- and backcross breeding. For a certain map location the function is
y_ij = m_j + e_ij
where y_ij is the value of the trait for the animal i, m_j is the mean effect of animals with genotype j, and e_ij is random error (Anderson, McRae, Visscher  2006).

Pleiotrophy:  One gene influences many phenotypic ("visible") traits. Examples of phenotypic traits are color and height.

QTL-mapping: Mapping of quantitative trait loci has four phases:

1. Measuring the quantitative traits to be researched
2. Analyzing the genotype of the animals to be researched
3. Building the connection maps
4. Finding statistically significant connections between desired traits and connection map

Mapping can be done either by studying a population or by cross-breeding two different lines of animals, and often inbred lines are used.

PS. Lastly, this is precisely my problem when studying:

Cramming for an exam on biotech

Although the lecturer said it'll be an exam with four essays, it can't hurt to clarify some of the key terms and themes concerning the basics of farm animal biotechnology. Ethical concerns and welfare issues are omitted - once I get started on that, there's no end to the rant.

Cloning: The practice of creating an animal from only one other animal of the same species. A cell sample, from skin or fur for example, is taken from the animal to be cloned. The nucleus is removed from one mature cell, and inserted into an egg cell, from which the nucleus has also been removed. The egg cell, now containing the genes of the animal to be cloned, is implanted on a female animal, which will eventually give birth to the cloned animal.

Genectic connection: For example, we might know that animals resistent to disease X have a mutation Y in their genome, while animals who suffer from X do not. Thus, even if it's not known which genes cause the resistance, the mutation Y can be used to  identify animals with X-resistance. Connections are deduced from the DNA of hundreds or preferably thousands of animals using statistical and genome analyzing tools.

Genetic marker: A piece of DNA, usually a microsatellite, which is connected to a specific trait. The marker can be either in a non-protein coding DNA or a part of the actual gene, in which case it is called the candidate gene. Markers are used to map genomes and to find genetic connections. Markers are not genes, and usually not active DNA at all. They can be thought of as genetic landmarks.

Marker assisted selection (MAS): The use of genetic markers linked to desired genes in breeding programmes. Animals can be selected to breeding based on traits which cannot be evaluated from the animal itself (like milk fat percentage from a bull) or while the animal is alive (such as pigs' carcass quality). When breeding choices are made from young animals and without genetic information, a male (like a popular dog) may pass on a serious disease to several offspring, before the male's sickness becomes known. MAS can be used to prevent this.

Microsatellite: A microsatellite is a short strand of DNA, which has a repetitive sequence of 2-4 bases, for example CACACACACACA. They are most often found in non-coding DNA. Microsatellites are found commonly on animal (including human) genome, and since they have a high polymorphism rate, they are often used in confirming descency and as genetic markers. Also known as STR or SSR for short tandem repeat or simple sequence repeat. Up to 30 % of a human genome consists of microsatellites, dinucleotine repeats being the most common.

Single-nucleotide polymorphism (SNP): SNP, pronounced snip, is a polymorphism of one nucleotide in DNA. A transition is when a purine is polymorphed to a purine or a pyriminide to a pyriminide (A to C, C to A, T to G, G to T). Transversion occurs when a purine is polymorphed to a pyriminide or vice versa. Both are explained clearly by Steven Carr.

Transgenesis: Inserting genes or other DNA material into a foreign cell. The original DNA can be taken from an animal of same or different species, and it may or may not be genetically modified. DNA can also be artificially created, and then inserted to the target cell. Transgenesis aims either at adding new properties, changing current properties or removing properties from the target.

Qualitative trait: A yes-or-no -trait, animal either has it or doesn't. Often an unwanted and monogenic trait, like a disease or bad meat quality. Whether a cow has horns or not is a qualitative, monogenic trait.

Quantitative trait: A trait, which is affected by several genes, possibly in several chromosomes. A polygenic trait, and often positive, such as milk yield in cows or large litter size in sows. A quantitative trait is described with a pluralistic variable like height, color, birth weight, milk yield or fat percentage. The color of a horse is defined by over 20 genes.


QTL/ETL: Quantitative trait locus / economic trait locus. Used to refer to a gene or a genetic marker related to a quantitative (polygenic) trait. Thus one gene solely responsible for a trait is not a QTL. QTLs for animals can be searched for example from Animalgenome.org.