• For the FRCS (Tr&Orth) exam genetics can be a rather awkward and annoying subject. It is generally a C list topic that only occasionally pitches up in the viva exam. Viva questions can be complicated to understand without a background knowledge of the basics of molecular biology. Finally, as orthopaedic surgeons we rarely use genetics in our daily clinical practice.
• However, it is important to have a basic understanding of molecular genetics in order to negotiate your way through any viva questions that may come your way. Genetics is at the cutting edge of new developments in medicine, and knowledge of the structure and function of genes is important to enable clinicians to identify and treat genetic diseases.
• Chromosomes are string-like structures made up of tightly coiled DNA around proteins called histones. The word chromosome is derived from the Greek words “chromo” meaning colour and “soma” meaning body (named so because they become strongly stained when colourful dyes are applied to them).

Figure 1. Structure of chromosome 

• The short arm of the chromosome is called p arm (from French: petit), the long arm the q arm and the constriction point between the two arms is the centromere.
• The tip of each arm is called the telomere.
• The position of the centromere is constant for a given chromosome and three subtypes are identified on the basis of the position of the position of the centromere:
• Metacentric centromere in the middle of the chromosome.
• Acrocentric centromere close to one end.
• Submetacentric intermediate position of centromere.
• A standardised numbering system is used for bands seen with G banding and this permits accurate description of breakpoints in chromosome rearrangements and is useful for describing the location of genes in the chromosomal map.
• There are 46 chromosomes in every nucleated, human cell, 22 pairs of autosomes and two sex chromosomes (male XY, female XX).
• Chromosomes are numbered according to their size, with chromosome 1 the largest, containing approximately 250 million base pairs of DNA, and chromosome 22 the smallest, containing approximately 40 million base pairs.

Point mutation

• A change in one base of the DNA sequence.

Substitution

• When one base or nucleotide in a sequence is replaced with another.

Insertion

• Where a base is added to the sequence.

Deletion

• Where a base is deleted from the sequence.

Inversion

• Where a segment of chromosome is reversed end to end.
• Occasionally a base substitution causes no change to a protein (silent mutation) but sometimes it can result in a change that can dramatically affect the function of a protein. The classic example is sickle cell disease in that only one DNA nucleotide difference out of a sequence of 438 results in the trait.
• A base substitution is often less harmful than a base deletion or insertion.
• mRNA is read as a series of triplets, adding or subtracting nucleotides may alter the triplet groupings of the genetic message. All the nucleotides that are “downstream” of the mutation will be regrouped into different codons. These new codons code for different amino acids that usually results in a different, and typically non-working, protein.

Figure 2. There are two broad categories of gene mutation: base substitution and base insertion (or deletion). The effect on the resulting polypeptide is shown here, following substitution (a) and deletion (b).

Gene duplications

• There is an increase in the number of copies of a gene.
• Deletion of large regions of a chromosome.
• Movements of sections of DNA from one location to another.

• Nucleic acid is the carrier of genetic information. There are two main types of nucleic acid, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), which each consist of a sugar-phosphate backbone with projecting nitrogen bases.
• The nitrogen bases are of two types, purines and pyrimidines.
• In DNA, there are two purine bases, adenine (A) and guanine (G) and two pyrimidine bases, thymine (T) and cytosine(C).
• RNA also contains A, G and C, but contains uracil (U) in place of T.
• In DNA the sugar is deoxyribose, whereas in RNA it is ribose.
• The nitrogenous bases are attached to the 1¢ (one prime) position of each sugar, and the phosphatelinks 3¢ and 5¢ hydroxyl groups.
• Each unit of purine or pyrimidine base together with the attached sugar and phosphate group is called a nucleotide.

Figure 3. DNA Structure

• A molecule of DNA is composed of two nucleotide chains that are coiled clockwise around one another to form a double helix with 10 nucleotides per complete turn of DNA.
• The two chains run in opposite directions (i.e. 5¢ to 3¢ for one and 3¢ to 5¢ for the other and are held together by hydrogen bonds between A in one chain and T in the other or between G and C.
• As A:T and C:G pairing is obligatory, the parallel strands must be complementary to each other.
• The ratio of A to T is 1:1 and of G to C is likewise 1:1 (Chargaff’s rule).

Figure 4. Section of a nucleic acid molecule showing two different nucleotides and their three parts (base, phosphate group and sugar

• Nucleic acids have 2 main functions: the direction of protein synthesis and the transmission of this information from one generation to the next
• Proteins, whether structural components, enzymes, carrier molecules, hormones or receptors are all composed of a sequence of amino acids.
• Twenty major amino acids are known, and the sequence of these determines the form and function of the resulting proteins
• DNA encodes all proteins and the unit of DNA that contains the protein-coding sequence (together with the introns and the neighbouring untranslated regulatory sequences) is, by definition its gene.
• Each set of three DNA base pairs (triplet or codon) codes for an amino acid. As each base in the triplet may be any of the four types of nucleotide (A, G, C or T), this results in 4³ or 64 possible combinations or codons.
• All amino acids except methionine and tryptophan are encoded by more than one codon, hence the code is said to be degenerate.
• Three codons designate termination of a message and are called stop codons and one codon acts as a start signal for protein synthesis.

• A gene is a sequence of DNA that gives rise to (codes for) the synthesis of a specific molecule of RNA or protein.
• Data from the Human Genome Project suggest there are around 24,000 protein coding genes and 8500 RNA genes (see Ensembl website for latest info).
• Only 1.1% of the genome is actually protein coding DNA.
• Another 4% consists of gene regulatory sequences and RNA genes.
• A large portion of the non-coding DNA, around 20% of the genome, consists of introns and untranslated regions of genes in addition to other non-coding gene-related sequences such as pseudogenes.
• The majority of the non-coding DNA, around 75% of the genome is extragenic, and much of this DNA (55% of the genome) consists of repeating sequences.
• Orthopaedic genome – a term used to describe the component of the genome relevant to orthopaedics.

• Genes encode proteins and proteins dictate cell function.
• Each step in the flow of information from DNA to RNA to protein provides the cell with a potential control point for self-regulating its functions by adjusting the amount and type of proteins it manufactures.

Figure 5. An overview of the flow of information from DNA to protein.Both coding and noncoding regions of DNA are transcribed into mRNA. Some regions are removed (introns) during initial mRNA processing. The remaining exons are then spliced together, and the spliced mRNA molecule (red) is prepared for export out of the nucleus through addition of an endcap (sphere) and a polyA tail. Once in the cytoplasm, the mRNA can be used to construct a protein.

• The first stage in gene expression is the synthesis of RNA molecules.
• Like DNA, RNA molecules compromise a sugar-phosphate backbone with bases attached, but RNA contains uracil (U) instead of thymine. Most RNA is single stranded.
• RNA molecules are synthesised by a process of transcription during which the DNA double helix unwinds and an enzyme, RNA polymerase makes copies of one strand of the DNA (Figure 6).
• Three types of RNA are synthesised: (1) ribosomal RNA; (2) transfer RNA; and (3) messenger RNA.
• Messenger RNA leads to the synthesis of specific proteins.
• Each gene that gives rise to mRNA is responsible for the synthesis of one, or a limited number of, related proteins. The genetic information responsible for the specificity of protein synthesis resides in the precise sequence of purine and pyrimidine bases contained within the gene.

Figure 6.Transcription

• The next stage of protein synthesis occurs in the cytoplasm and is called translation. Each mRNA molecule becomes attached to one or more ribosomes. As the ribosome moves along the mRNA from the 5 to 3 end each codon is recognised by a matching tRNA which contributes its amino acid to the end of a new growing protein chain until a stop codon is reached.

• The coding sequences of the genes (exons) are interrupted by non-coding sequences (introns). After a gene has been transcribed into a complementary RNA copy, this transcript undergoes splicing to remove introns.
• Different exons from the same gene can be spliced together, yielding different mRNA molecules and, in time, different proteins. Splicing and other chemical changes to the RNA are known collectively as RNA processing.

• Processed mRNA molecules leave the nucleus and engage the ribosomes, where protein synthesis occurs by a process known as translation.
• During translation, the nucleotide sequence within each mRNA molecule is read sequentially. Each run of three nucleotides, known as a codon, specifies the insertion of a particular amino acid into the growing peptide chain. A single initiation codon, AUG (which codes for methionine), begins the process of translation.
• TRNA brings the amino acids to their place within the protein.
• Other codons, stop codons (UGA, UAA, and UAG), terminate elongation of the protein chain, which is then released from the ribosome.

Figure 7.Translation

• New protein molecules may undergo a variety of posttranslational modifications, including specific cleavage by proteinases; the covalent addition of various moieties such as phosphate, lipid, and sugar molecules; as well as additional chemical modification of certain amino acids.
• Therefore by a combination of mRNA splicing and posttranslational modification, a single gene may give rise to several different proteins.

• These genes play important roles in maintaining homeostatic cell physiology.
• They are expressed at a fixed rate, irrespective of the cell condition.

• These genes are expressed only in certain tissues or cell types and/or at certain times only as needed.
• Their amount may increase or decrease with respect to their basal level in different conditions.
• Their structure is relatively complicated.

• Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.

• Regulation of gene expression includes a wide variety of mechanisms that are used by cells to increase or decrease the production of specific gene products.
• Any step of the gene's expression may be modulated, from DNA-RNA transcription to the posttranslational modification of a protein.
• A promotor is a sequence of DNA that defines where transcription of a gene by RNA polymerase begins. Promotor sequences are usually located upstream and often contain a sequence called the TATA box (Figure 8).

Figure 8. Located approximately 25-35 base pairs upstream of the transcription unit, the TATA box is a highly conserved sequence that works to help position RNA polymerase II during the iniation of transcription.

• An enhancer is a short region of DNA that when bound by specific proteins called transcription factors, enhance the transcription of an associated gene (Figure 9).
• Enhancers can be located upstream, downstream or within the gene that is transcribed.
• Most enhancers are active only in specific cell types and therefore play a major role in regulating tissue specificity of gene expression.
• Each enhancer has its own transcription factor that it binds to.
• Silencers are similar sequence elements that, in contrast inhibit transcription of the associated gene.

Figure 9. Action of an enhancer.An enhancer binding protein has 2 binding sites(1)Binds DNA (2)Binds the transcription factors that are bound to the promotor

• The expression of many genes is modulated in response to external stimuli, such as growth factors and hormones.
• In most cases, the process is initiated when a growth factor or hormone binds to a specific cell-surface receptor. This triggers the process of signal transduction, during which the occupied receptor initiates a chain of intracellular, biochemical changes that result in altered gene expression.

• This involves isolating fragments of DNA and replicating them.
• Although entire genes can be cloned, these are very large and a simpler process involves generating complimentary DNA (cDNA) that lacks introns.
• This is performed by using the enzyme reverse transcriptase (RT) that copies mRNA into cDNA.
• The cDNA generated by reverse transcriptase can then be spliced into plasmids, which are circular molecules of DNA that exist within bacterial cells.
• Several hundred copies of plasmids per bacterium can be present and they are replicated as the bacterial host divide.

• It is now possible to produce large amounts of therapeutic protein for clinical use.
• The cDNA encoding the protein of interest is isolated and spliced into an expression plasmid.
• The plasmid is transferred into host cells that then express the transgene of interest.
• Examples of recombinant protein synthesised include erythroprotein, BMP-2 and BMP-7.

• Single nucleotide polymorphisms (SNPs) are a type of polymorphism involving variation of a single base pair.
• They are the most common type of genetic variation among people.
• Each SNP represents a difference in a single nucleotide.
• Most SNPs have no effect on health as they generally occur in non-coding gene regions; however, they can act as biological markers to locate genes that are associated with disease.
• When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting a gene’s function.
• Researchers have found SNPs that may help predict an individual’s response to certain drugs, susceptibility to environmental factors such as toxins, and risk of developing particular diseases.
• SNPs can also be used to track the inheritance of disease genes within families.

Figure 10. SNP’s.

• Gel electrophoresis is a technique used in laboratories to separate charged molecules such as DNA and RNA according to their size.
• An electric current is applied across the gel.
• The gel consists of a permeable matrix through which molecules can travel when an electric current is passed across it.
• A molecule with a negative charge will migrate towards the positive end.
• Smaller molecules migrate through the gel more quickly and travel further than larger fragments that migrate more slowly and travel shorter distances. Therefore the molecules are separated by size.
• The use of dyes, fluorescent tags or radioactive labels enables the DNA on the gel to be visualised after they have been separated.
• A DNA marker with fragments of known lengths is usually run through the gel at the same time as the samples. By comparing the bands of the DNA samples with those from the DNA marker it is possible to work out the approximate length of the DNA fragments in the samples.
• Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose depends on the size of DNA being analysed.
• The gel is placed into an electrophoresis tank and electrophoresis buffer is poured into the tank. The buffer conducts the electric current. The type of buffer depends on the approximate size of the DNA fragments in the sample.

• These enzymes cleave DNA at specific recognition nucleotide sequences known as restriction sites along the molecule.
• Biological scissors.
• Found naturally in bacteria
• “Immune system” of bacteria:

1. Protect bacteria against intruding DNA from other organisms (phages, other bacteria).
2. Recognise short nucleotide sequences in the foreign DNA.
3. Cut covalent phosphodiester bonds of both strands of DNA, rendering foreign DNA harmless.

• The rarer the site it recognises, the smaller the number of pieces produced by the given restriction.
• Used in any process that involves manipulating, analysing, and creating new combinations of DNA sequences.
• Uses include DNA cloning, hereditary disease diagnosis, paternity testing, genomics (e.g. the human genome project) and epigenetics.

Figure 11. Restriction enzymes act like biological scissors

• This involves taking DNA from two different sources and combining that DNA into a single molecule.
• The artificially created DNA is then reproduced by cloning.
• Six steps are involved in the process:

1. Isolating
2. Cutting (cleavage)

This involves cleavage of DNA to generate fragments of defined length, or with specific endpoints using restriction enzymes (see above). The DNA fragment of interest is called insert DNA.

1. Joining (ligation of DNA fragments)

A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and then ligating the insert DNA to vector DNA (recombinant DNA or chimeric DNA). DNA fragments are usually joined using DNA ligase.

2. Transforming

The recombinant DNA molecule is introduced into a compatible host cell where it can replicate. The direct uptake of foreign DNA by a host cell is called genetic transformation. Recombinant DNA can be packaged into virus particles and transferred to host cells by transfection.

3. Cloning

Cloning vectors allow replication and expression of recombinant DNA in host cells.

4. Selecting

This involves the identification of host cells that contain the recombinant DNA of interest.

• PCR is a molecular biology technique for enzymatically replicating small sections of DNA or a gene.
• The technique allows a small amount of DNA to be amplified many times in an exponential manner and generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA (Figure 13).
• Five basic components are required to set up a PCR.

1. The DNA template to be copied.
2. Two primers-short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the section of DNA to be copied.
3. DNA nucleotide bases (A, C, G and T).
4. Taq polymerase enzyme to add in the new DNA bases.
5. Buffer to ensure the correct chemical environment for the DNA polymerase reaction.

• PCR involves a process of heating and cooling called thermal cycling.
• The PCR reaction is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction.
• There are three main stages:

1. Denaturing

When the double stranded template DNA is heated to separate it into two single strands.

1. Annealing

After separating the DNA strands, the temperature is lowered to enable DNA primers to attached to the single strand (template) DNA.

2. Extension

The temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

Denaturing stage

• The high temperature causes the hydrogen bonds between the bases in the two strands of template DNA to break and the two strands to separate.

Annealing stage

• The primers attach to a specific location on the single stranded template DNA by way of hydrogen bonding.
• Primers are single strands of DNA or RNA sequence that are around 20–30 bases in length.
• The primers are designed to be complementary in sequence to short sections of DNA on each end of the sequence to be copied.
• Primers serve as the starting point for DNA synthesis. Only once the primer has bound can the polymerase enzyme attach and start producing the new complementary strand of DNA from the loose DNA bases.

Extending stage

• The heat is increased to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases.
• The end result is a brand new strand of DNA and a double stranded molecule of DNA.

Taq DNA polymerase

• This is a thermal stable enzyme isolated from thermophilic bacteria. This enzyme commonly synthesises DNA in one direction 3¢ to 5¢.

Applications of PCR

• Molecular biological research:

• Gene screening analysis (looking for a gene) and DNA cloning (copying particular DNA sequences).

• Genetic mapping studies e.g. human genome project.
• Clinical and diagnostic uses:

• Screening and diagnosis of cancer (detects mutations of oncogenes), genetic disorders.

• Genetic identification and DNA typing:

• Sex determination of prenatal cells.

• Identification of trace amounts of DNA:

• Detection of contamination of foodstuff, pork in beef, etc.

• RT–PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. The newly synthesised cDNA can be amplified using traditional PCR.

Figure 14. In reverse transcription a single-stranded complementary DNA (cDNA) is synthesized based on the sequence of the target RNA (Figure 1). Reverse transcription requires an oligonucleotide primer to initiate the reaction and an enzyme, reverse transcriptase. The cDNA can be further used in PCR for the amplification.

• DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. Southern blotting could be used to locate a particular gene within an entire genome.
• Southern blotting results in transfer of DNA molecules, usually restriction fragments, from an electrophoresis gel to a nitrocellulose or nylon sheet (referred to as a “membrane”), in such a way that the DNA banding pattern present in the gel is reproduced on the membrane.
• During transfer or as a result of subsequent treatment, the DNA becomes immobilised on the membrane and can be used as a substrate for hybridisation analysis with labelled DNA or RNA probes that specifically target individual restriction fragments in the blotted DNA.
• In essence, Southern blotting is a method for “detection of a specific restriction fragment against a background of many other restriction fragments.”

• This is a technique used in molecular biology research to study gene expression in a sample, through detection of RNA (or isolated messenger RNA).
• With Northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions.
• Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridisation probe complementary to part of or the entire target sequence.
• The blot is incubated with a probe which is single-stranded DNA. This probe will form base pairs with its complementary RNA sequence and bind to form a double-stranded RNA-DNA molecule. The probe is either radioactive or has an enzyme bound to it.

• A method used to detect and analyse proteins.
• This involves transferring, or blotting, proteins separated by electrophoresis from the gel to a membrane.
• To visualise the protein of interest the membrane is commonly first probed using a primary protein-specific antibody followed by a labelled secondary antibody used for detection. An image is taken of the membrane and the result is analysed.

• This uses specific restriction enzymes to cut an unknown segment of DNA at short, known base sequences called restriction sites.
• Restriction enzymes always cut DNA at a specific sequence of DNA (restriction site). For example, the restriction enzyme EcoRI (taken from E. coli always cuts at the sequence GAATTC/CTTAAG. Therefore if we use EcoRI to cut the DNA we know that the DNA sequence either side of the cut will be AATT (see Figure 15). 
• A restriction map shows all the locations of that particular restriction site (GAATTC) throughout the genome.

Figure 15. Diagram demonstrating the restriction site for the restriction enzyme EcoRI. Restriction enzymes always cut DNA at a specific sequence of DNA

• A plasmid is a small extrachromosomal, circular piece of DNA that replicates independently of the host DNA.

• Gene therapy is when DNA is introduced into a patient to treat a genetic disease. The new DNA usually contains a functional gene to correct the effects of a disease causing mutation.
• It was anticipated that gene therapy would provide treatments for genetic conditions such as muscular dystrophy and cystic fibrosis but so far, however, it has had only limited success in treating human disease.
• There are two different types of gene therapy.

Somatic gene therapy

• Transfer of a section of DNA to any cell in the body that does not produce sperm or eggs. Effects of gene therapy will not be passed onto the patient’s children.

Germline gene therapy

• Transfer of a section of DNA to cells that produce eggs or sperm. Effects of gene therapy will be passed into the patient’s children and subsequent generation.

• This is used to treat diseases caused by a mutation that stops a gene from producing a functioning product, such as a protein.
• This therapy adds DNA containing a functional version of the lost gene back into the cell.
• The new gene produces a functioning product at sufficient levels to replace the protein that was originally missing.
• This is only successful if the effects of the disease are reversible or have not resulted in lasting damage to the body.
• For example, this can be used to treat loss of function disorders such as cystic fibrosis by introducing a functional copy of the gene to correct the disease.

Figure 16. Gene augmentation therapy

• Possible uses include the treatment of infectious diseases, cancer and inherited diseases caused by inappropriate disease activity.
• The aim is to introduce a gene whose product either:

1. Inhibits the expression of another gene.
2. Interferes with the activity of the product of another gene.

• The basis of this therapy is to eliminate the activity of a gene that encourages the growth of disease-related cells.
• Certain cancers may be caused by the overactivity of an oncogene. By eliminating the activity of that oncogene through gene inhibition this prevents further cell growth and cancer spread.

Figure 17. Gene inhibition therapy

• This is suitable for diseases such as cancer that can be treated by destroying certain groups of cells.
• The aim is to insert DNA into a diseased cell that causes that cell to die.
• This can be achieved in one of two ways:

1. The inserted DNA contains a “suicide” gene that produces a highly toxic product which kills the diseased cell.
2. The inserted DNA causes expression of a protein that marks the cells so that the diseased cells are attacked by the body’s natural immune system.

• A section of DNA/gene containing instructions for making a useful protein is packaged within a vector, usually a virus, bacterium.
• The vector acts as a vehicle to carry the new DNA into the cells of a patient with a genetic disease.
• Once inside the cells of the patient, the DNA/gene is expressed by the cell’s normal machinery leading to production of the therapeutic protein and treatment of the patient’s disease.
• It is essential with this method that the inserted DNA is targeted appropriately to avoid the death of cells that are functioning normally.

Figure 18. Killing of specific cells.

• A vector is a DNA molecule used as a vehicle to carry foreign genetic material artificially into another cell where it can be replicated and/or expressed.
• A vector can be a virus, bacterium or plasmid.
• A section of DNA/gene containing instructions for making a protein is packaged within a vector.
• The vector acts as a vehicle to carry the new DNA into the cells of a patient with a genetic disease.
• Once inside the cells of the patient, the DNA/gene is expressed by the cell’s normal machinery leading to production of the therapeutic protein and treatment of the patient’s disease.

Expression vectors

• These vectors are for the expression of the transgene in the target cell. They would include elements for translation of a protein, such as a promotor sequence that drives expression of the transgene, a ribosome binding site, stop and start codons.

Transcription vectors

• These vectors are only capable of being translated but not transcribed.

Figure 19. DNA transfer

• It is vital the new gene targets the correct cell and is then switched on.
• On occasions the immune system can be activated and this could cause serious illness and death. The risks of immune system triggering are reduced by using the lowest dose of a virus that is effective and using vectors that are less likely to trigger an immune response.
• Ideally, a new gene introduced by gene therapy will integrate itself into the genome of the patient and not disrupt the function of other genes. Disrupting important genes in target cells may lead to tumour formation or death. Safer ways have been developed to introduce genes which include newer vectors that target DNA integration into specific safe places in the genome.
• Once the gene is switched on it must remain on, cells have a habit of shutting down genes that are either too active or exhibit unusual behaviour.
• The gene may be incorporated into the wrong cell. Improper targeting is potentially harmful.
• If the gene is incorporated into a patient’s germline it may be passed on to future offspring. This could lead to heritable alterations in the genome that could be passed on to future generations, rather than the effects being confined to one person. This violates the Weismann barrier principle.
• The high cost of developing gene therapy for rare diseases may make this research unappealing for pharmaceutical companies.

• Weismann barrier principle: The principle that hereditary information moves only from genes to body cells, and never in reverse.
• Transgene: A transgene is a gene or genetic material that has been transferred naturally or by a number of genetic engineering techniques from one organism to another.
• Insert: An insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination.