recombinant dna technology

• complementary DNA can be made using reverse transcriptase. reverse transcriptase is an enzyme found in retro viruses which catalyses the formation of double stranded DNA from single stranded RNA. Reverse transcriptase synthesises a cDNA strand, which can be used as a template for DNA polymerase to form double stranded DNA. This is useful as we can make versions of DNA to act as genes, by extracting mRNA from a cell where that gene is being expressed.
• restriction endonucleases are used to cut DNA into fragments. Bacteria produce enzymes that can cut viral DNA at a specific sequence of bases (A recognition sequence). This can happen between a complementary base pair, leaving a blunt end. While others cut in a staggered fashion, leaving sticky ends where a sequence of unpaired bases remains. If read left to right this is a palindrome. SO the recognition sequence for sticky ends is known as a six bp palindromic sequence.
• Sticky ends are important as if two different DNA molecules are cut with the same restriction endonucleases, their sticky ends will be complementary. So DNA fragments can be temporarily joined together until more permanent covalent bonds can be formed between the sugar phosphate backbone by DNA ligase , forming recombinant DNA
• Once a fragment of DNA with the desired gene has been obtained, it can be cloned in vivo by transferring the fragments to a host cell using a vector. A promoter and terminator region must be added to the fragment, so the RNA polymerase can attach to the gene allowing it to be expressed, and detach at the appropriate point. Plasmids are the most common vector. the same restriction endonuclease used to cut the fragment cuts the plasmid, so it is complementary. Once the plasmid is opened up, the fragment can become incorporated, with this incorporation made permanent by DNA ligase
• A modified vector (DNA incorporated) is reintroduced into bacterial cells via a transformation. The vector and Bacteria are mixed together in a medium containing calcium ions and exposed to temperature changes, making the bacterial membrane permeable and allowing the plasmid to pass into the cytoplasm. Not all bacteria that undergo transformation will possess the desired gene for the desired protein. As, only a few bacterial cells take up the plasmid, some plasmids will have closed up again without incorporating the plasmid, sometimes the DNA fragment ends join forming its own plasmid.
• gene markers are used to know if bacteria have taken up the recombinant DNA, with marker genes like antibiotic resistance genes, fluorescent marker, and enzyme marker. These determine if the transformation process has been successful, and the bacteria with the plasmid can be detected and seperated.
• antibiotic resistant gene markers: involves replica plating. The antibiotic resistant gene in the plasmid is cut open to incorporate the desired gene. If the recombinant DNA is incorporated, the resistant gene cannot be expressed, and the bacteria will no longer be antibiotic resistance. Growing the bacteria on antibiotic plates will show that the destroyed bacteria have taken up the gene, but replica plating must be used or you will destroy the colony of bacteria you want
• fluorescent gene markers: a gene from a jellyfish is transferred to a plasmid, the gene produces a green fluorescent protein. The recombinant DNA gene is inserted into the fluorescent protein gene, so the bacteria which glow have not incorporated the desired gene. This does not require replica plating as you don't kill your desired bacteria colony during testing
• enzyme gene markers: add a gene which produces the lactase enzyme to the plasmid, this will turn a particular colourless substrate blue. Insert the desired gene into the gene for the lactase enzyme. If the bacteria still produces lactase and turns the colourless substrate blue, then it does not contain the desired gene.
• The polymerase chain reaction (PCR) is a method for amplifying a desired DNA molecule. It is carried out in cycles, with each cycle doubling the number of DNA molecules present. It is rapid and efficient. It is carried out in a thermocycler, with nucleotides, primers, DNA polymerase and the DNA fragment desired present.
• PCR is carried out in a thermocycler in three stages. First the DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler. The temperature is increased to 95 degrees, hydrogen bonds between complementary base pairs are broken and the two DNA strands separate. Then annealing primers are added, and the mixture is cooled to 55 degrees, the primers anneal to their complementary bases at the end of the DNA fragment. (prevent strands rejoining and allows DNA polymerase to work as it can only ass nucleotides to the end of an existing chain).
• Once the primers have annealed to the end DNA fragment in PCR, the temperature is increased to 72 degrees ,the optimum temperature for DNA polymerase to synthesise a new DNA strand, and a new strand of DNA is synthesised from the primer. The process is repeated by subjecting the new strands to the temperature cycle again. Many copies of the desired DNA fragment can be made very quickly
• PCR - in vitro, Vectors - in vivo
• advantages of in vitro cloning: - It's extremely rapid so billions of copies of a gene can be made in a few hours, valuable when only a small amount of DNA available from a crime scene, quickly increased with little loss of time before forensic analysis can take place. (will also amplify other contaminating DNA). It does not require living cells, only the DNA base sequence to be amplified is needed, so not complex culturing techniques are needed
• advantages of in vivo cloning: - useful when we wish to introduce a gene into another organism (gene therapy). Almost no risk of contamination, only DNA cut with the restriction endonuclease the plasmid was cut with is complementary to the plasmids sticky ends. So no contaminating DNA could be taken up and amplified. It's very accurate, has a lesser rate of mutation than in vitro cloning. It cuts out specific genes, very precise procedure with many copies of the specific gene, not the whole DNA sample, made. The transformed bacteria can produce proteins for commercial use
• A DNA probe is a short, single stranded DNA molecule designed to be complementary to a sequence that needs to be detected. The probe is radioactively or fluorescently labelled so it can be detected once it has bound to the desired sequence. The probe is made to have a complementary base sequence to the part of the DNA making up the desired allele, the double stranded DNA is treated to separate the strands, the probe is mixed with the strands and binds, DNA hybridisation, the site at which the probe binds can then be identified by the marker.
• DNA hybridisation takes place when a section of DNA is combined with a single stranded section of DNA with complementary bases. First the DNA must be heated until the strands separate, then the single stranded DNA molecules are added. As the DNA cools the complementary bases anneal, recombining complementary strands. The complementary sections of DNA are just as likely to anneal to the DNA strands as the complementary strand
• locating specific alleles of genes: determine the sequence of nucleotide bases present on the allele we wish to locate. Produce fragment of DNA complementary to this sequence. Amplify fragment in PCR. attach marker to form DNA probe. Heat DNA suspected to have DNA sequence you want to find, separating strands, cool in mixture of many DNA probes. If dna contains complementary base sequence it will likely bind to the probes, so when washed clean of unattached probes any remaining hybridised DNA will be labelled by the probe and identifiable
• DNA probes can be used to screen for heritable conditions and health risks. Many genetic disorders result from gene mutations, is these result in a recessive allele, some people with family history of diseases like sickle cell anaemia may be carriers. They could pass this disease onto their children if their partner is also a carrier. By screening, they can obtain advice from a genetic counsellor about potential implications for their children.
• DNA probes can be fixed to a glass slide, each with a differing nucleotide base sequence complementary to a different sequences associated with diseases. A DNA sample can be added and multiple diseases can be tested for at once. DNA probes in genetic screening can also detect oncogenes and mutated tumour suppressor genes. Some people inherit one mutated tumour suppressor gene ( 2 for tumour formation) and are at a higher risk of cancer. By knowing they have this mutation they can alter there lifestyle and come in for more regular testing so any cancer is detected earlier
• Genetic counselling involves giving people advice and information to enable people to make personal decisions about themselves and their offspring. Genetic counsellors research family history of inherited disease to advise parents on the likelihood of it arising in their children. They also advise parents on the potential effects of the disease for their child, if they do inherit it, and the emotional, medical, social, and economic impacts. It can also make couples aware of potential screening tests for their embryos if they have IVF
• Genetic counselling is closely linked to genetic screening. Results provide the genetic counsellor with a basis for informed discussion. About oncogene mutations and the type of cancer someone has and so the most effective treatment. Potential future cancer development, so they get tested more often so cancer is picked up earlier and prognosis is better
• Genetic fingerprinting is a technique that can detect differences in people's DNA, it uses non-coding regions of DNA called variable number tandem repeats. Every individual has a differing number and length of VNTRs, except identical twins, so they act like a fingerprint. The more closely related to individuals are, the more similar their VNTRs
• Gel electrophoresis is a way of separating DNA fragments based on their size. DNA fragments are placed in agar gel and a voltage is applied across it. As DNA is a negatively charged molecule and is attracted to the positive charge of the electric field. The resistance of the gel means that the larger fragments move slower. If DNA probes are attached to the fragments, with markers, they can be situated on the gel. Larger DNA fragments cannot be sequenced this way, they must first be cut into smaller fragments using restriction endonucleases
• genetic fingerprinting is sensitive, with only a tiny DNA sample needed. It is done in five stages. Extraction: DNA is separated from animal tissue and the rest of the cell, then amplified via PCR. Digestion: DNA cut into fragments with restriction endonucleases. Separation: DNA fragments separated according to size via gel electrophoresis. Then submerged in alkali to separate the two strands. Hybridisation: DNA probes bind with VNTRs under specific conditions, with different probes binding to different sequences. Development: put x ray film on nylon membrane, becomes exposed locating probes
• DNA fingerprint results are compared between two samples. First they are visually checked, and if they appear to be a match they are passed through an automated scanning machine which calculates the length of DNA fragments from the bands. The odds are then calculated of someone else having an identical fingerprint. The closer the match between the 2 patterns the higher the chance they come from the same person
• Genetic fingerprinting has many uses: Genetic relationships and variability - paternity tests and determining genetic variability within a population. Forensic science- DNA left at crime scenes can determine wether a suspect was present ( must consider it may have been a close relative of the suspect, left on another occasion, have been contaminated after the crime, or someone elses DNA might match the suspects). Medical diagnosis- diagnose diseases like huntington's, or the nature of microbial infection. plant and animal breeding - prevent undesirable or inbreeding in zoos or farms