Gene cloning is a fundamental process in genetic biotechnology, employing techniques of molecular genetics to isolate and build copies, or clones, of genetic material of interest, often a specific gene or other short sequence of DNA. In brief, the general procedure to clone a gene is to isolate the specific sequence of DNA, insert it into a vector plasmid (circular DNA that acts as a carrier), introduce the gene-carrying plasmid into bacteria (transformation), then harness the molecular machinery of the bacteria to either replicate the gene of interest or produce its gene product.
Restriction enzymes are a class of enzymes which cut double-stranded DNA in a way that is particularly useful to isolating and handling a gene or sequence of interest.
First, each type of restriction enzyme works to cut at a specific location based on a unique nucleotide pattern, which it then identifies in the target DNA. Using a particular restriction enzyme on the target DNA will always produce the same DNA fragments because the cut location is pattern specific.
Second, the DNA fragments will have overhang on opposing strands at each cut site. (The restriction enzyme does not make a blunt cut across the DNA, but rather in a zigzag.) Because the cut was made according to a specific pattern, the overhanging nucleotides will base-pair perfectly with other ends cut by the same restriction enzyme. This allows the target fragment to be inserted into another DNA source treated by the same restriction enzyme (e.g. a plasmid).
Once two ends are annealed by base pairing, they then need to be sealed by DNA ligase.
A DNA library is a collection of DNA fragments that encode for the mRNA transcripts expressed by a source of interest, such as cells from a particular tissue or transcriptome under certain environmental conditions. Control of gene expression determines which sequences of DNA (e.g. within a genome) are transcribed and then spliced into mature mRNA for translation into a protein product. A sample mature mRNA is used to generate complimentary DNA (cDNA), which represents a DNA sequence that encodes for the mRNA of interest. This collection of cDNA fragments can then be stored in a vector (the DNA library) for sequencing or later expression.
cDNA is produced opposite the flow of the Central Dogma (DNA → RNA → polypeptide). Source mRNA is treated with the enzyme reverse transcriptase, which reads a strand of mRNA and generates a matching single-stranded DNA sequence. DNA Polymerase is used to build out the partner strand. The final product is a length of double-stranded DNA that can be transcribed into the mRNA of interest. Because the mRNA used would have already undergone splicing, the resulting cDNA will not have the sequences representing introns (or unselected exons from alternate splicing) and will only have the expressed exons.
Hybridization is a technique that harnesses the base-pairing of complimentary strands to ascertain the presence of particular mRNA transcripts in a sample by exposing the sample to known complimentary mRNA and measuring the amount of binding. The procedure can be performed with a number of different mRNA transcripts in a sample which is then applied over a microarray that contains complimentary mRNA in a collection of separate wells for binding the appropriate mRNA from the sample if present. Fluorescent labeling (a tag or probe) can be added to the sample mRNA ahead of time to aid in measuring the amount of binding taking place for each complimentary mRNA.
As mentioned above, the mRNA transcript from a gene of interest can be reverse transcribed into a cDNA fragment that contains only the exon sequences. This cDNA fragment can be introduced into a plasmid along with a gene for antibiotic resistance. This allows for isolation of only successfully transformed (took the plasmid) bacteria when exposed to the plasmid and then grown in the presence of the antibiotic. Along with resistance to the antibiotic that allows these bacteria to survive, the cells now also have the cDNA fragment of interest. The bacteria can then be grown to produce the protein product through their normal means of gene expression.
Polymerase chain reaction (PCR) is a procedure for producing copious amounts of a DNA fragment of interest. The initial sample of DNA is taken through a series of heating and cooling steps that denature the DNA, separating it into its two strands to allow for in vitro replication along each strand. By adding a DNA primer to begin replication in opposite directions at the end of each separated strand, a heat-resistant DNA polymerase (Taq polymerase) and nucleotides to build new DNA, each cycle produces another replication of the fragments created in the previous cycle, quickly creating a large amount of copies of the sample DNA.
Gel electrophoresis is a technique for separating and visualizing the presence of DNA fragments in a sample. The process works by applying a current across a gel that has samples of DNA injected into wells in the gel at one end. The gel is submersed in a buffer solution, and the DNA, holding a negative charge from its phosphate groups, will be pulled across the gel towards the positively charged anode (as an electrolytic cell, negative charge is flowing from this anode to a battery or power source, leaving it with a positive charge). Smaller fragments will move faster than larger fragments, thus separating the sample by fragment size into bands that have moved across the gel. Generally, samples are run adjacent to a known sample, a DNA ladder, that will produce bands at sized-determined distances for comparison and estimation of sample fragment sizes. Fragments of more base pairs will not have moved as much and will be higher in a gel analysis, whereas smaller fragments of lower base pairs will be towards the bottom of the gel, having travelled farther.
A Southern Blot provides further analysis of sample DNA fragments. A membrane is applied to a gel after electrophoresis, taking up the DNA fragments in the pattern created on the gel. A hybridization probe (known DNA fragment complimentary to sequence of interest) is then applied and finally washed away from the membrane. Any presence of the target sequence can then be identified by a tag (fluorescence, radioactivity, or chromogenic dye) now bound to the transferred samples. Thus a specific DNA sequence or gene can be identified as being present in the original sample.
DNA sequencing determines the nucleotide sequence in a sample of DNA. There are numerous techniques to perform sequencing with most beginning with an amplification of the DNA sample such as with PCR or use of a bacterial vector. The early successful chain-termination method involves in vitro replication with the introduction of labelled dideoxynucleotides (special As, Ts, Gs, and Cs that cannot be elongated from). These special types of nucleotides block the addition of more nucleotides, thus terminating the chain. By comparing the lengths of the (termination-shortened) fragments and identifying the nucleotide by its label, a sequence of the original sample DNA can be determined. A technique called shotgun sequencing involves creating random fragments from a large DNA molecule, individually sequencing the smaller fragments, then determining the sequence the entire original length by overlapping the fragments.
There are many studies that can be made of gene expression. Gene products can be the final translated polypeptides or can be of a type of non-coding RNA that have their own roles within the cell, generally for regulating transcript and translation. A transcriptome, the total of RNAs or exclusively mRNAs produced in a cell or tissue, reflects the products of gene expression. Additionally, specific protein products themselves can be detected using a technique similar to Southern blotting called a Western blot.
There are several approaches to determining gene function. A gene can be sequenced then compared to similar sequences in the genome to identify potential similarity with known gene products. Knockout models silence specific genes to test for loss of function or other phenotypic affects.
Stem cells are a cells with the characteristic that they have not yet undergone processes of differentiation, thereby maintaining an unspecialized nature. Specialization can be determined by permanent structural changes to the genome such as DNA methylation to silence specific genes. More specialized cells and tissues can then be derived from these cells during development and growth. Cells with the least specialization are called pluripotent stem cells, acknowledging a potential to transform into any number of tissue types. Other stem cell types include multipotent and hematopoietic stem cells. Research into induced pluripotent stem cells looks into the reverting of somatic (specialized) cells back to pluripotency.
Genetic biotechnology has numerous applications for engineering and medicine.
Recombinant DNA technology has been used to mass produce protein products such as insulin and human growth factor. Genome sequencing and gene functional analysis have also impacted genetic counseling and diagnosis.
Efforts have been made to target specific disease causing mutations using gene therapy.
Recombinant DNA technology can also be applied to vaccine production and to identify pharmaceutical targets.
Analysis of variations in non-coding regions of the genome (e.g. short tandem repeats) and mitochondrial DNA can be used to identify individuals and their genetic relationships.
Harnessing the gene products of microorganisms can treat sewage or clean up chemical spills.
Recombinant DNA has been used to introduce genes into crops for resistance to herbicides and to delay ripening during transportation.
Recombinant DNA technologies involve the risk of undesirable outcomes stemming from the same genetic recombination power that has driven evolution. The genetic basis of cancer is suggestive of the potential for harm in employing genetic therapies and technologies. Safety guidelines have been developed to protect researchers and the public. Ethical issues continue around aspects of predictive genetic markers, germline alteration, privacy, and socio-economical access.
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