Topic 2.5 Application of Reproduction & Genetics Flashcards
a) The Human Genome Project - What were the 5 aims to fulfill?
b) What 3 major things did they find?
a) The Human Genome Project had FIVE aims to fulfill:
1. Identify all genes in the human genome & identify/find which chromosomes each gene is on.
2. Determine the sequence of the 3 bn base pairs (the 2 nitrogen-containing bases that pair together to form the structure of DNA) in the human DNA and store this information on a database.
3. Improve tools for analysis
4. Transfer reated technologies to the private sector - medical innovation.
5. Address ethical, legal and socail concerns.
b) They found:
1. Humans have 20,500 genes (less than expected initially)
2. More repeated segments of DNA (again, wasn’t expected initially)
3. Fewer than 7% of the families of proteins were SPECIFIC to vertabrates, this small number highlighted how interconnected living organisms are.
a) The 100K Genome Project: Subsequent to the success of the HGP, the 100K project was launched and had the aim of using WHAT (and explain what this “what” is) to sequence 100,000 WHAT from SPECIFICLLY…WHICH type of people…who have WHAT and other rare WHAT?
b) The aims of the 100K project were? There are 4.
c) In what cenario would genetic screening be helpful to a doctor?
a) Subsequent to the success of the HUMAN GENOME PROJECT. The 100K project was aimed at using NGS (Next Generation Sequencing - these are faster and cheaper techniques used to sequence base pairs. It’s NOT like sanger sequencing which is a LOOONG method) to sequence 100,000 genomes from SPECIFICALLY NHS patients with CANCER and other rare diseases.
b) Aims:
1. Create ETHICAL, transparent programmes based on consent of patients.
2. Set up a genomics service run by the NHS with the aim of benefitting the public
3. Enable the development of medical and scientific research
4. Kick-start the genomics industry
c) The diagnosis can be improved as well as the treatment of the disease. Doctors can combine sequencing data with patient records to piece together a diagnosis.
a) Sanger Sequencing is a method of seqencing DNA (DNA sequencing is the process of determining the exact number of nucleotides witihin a molecule of DNA. This technique copies DNA _____ ______, making __________ of the DNA of varying ________ up to ___ bases in length. A fluorescent ‘chain __________’ nucleotide marks the ____ of each of the fragments, with 4 distinct colours for each of the 4 ______. The fragments are then loaded onto a ___ ______________ _______. ________ fragments move quicker and ________ fragments less quickly. So they therefore arrange themselves in ____ order. The colours then showcase the order of the bases.
b) What is the other name of Sanger Sequencing?
c) What is electrophoresis?
a) Sanger Sequencing is a method of seqencing DNA (DNA sequencing is the process of determining the exact number of nucleotides witihin a molecule of DNA. This technique copies DNA MANY times, making fragements of the DNA of varying lengths up to 900 bases in length. A fluorescent ‘chain terminator’ nucleotide marks the END of each of the fragments, with 4 distinct colours for each of the 4 bases. The fragments are then loaded onto a gel electropheresis plate. Smaller fragments move quicker and larger fragments less quickly. So they therefore arrange themselves in size order. The colours then showcase the order of the bases.
b) Also called ‘chain termination method’.
c) Electrophoresis = A technique that seperates molecules on the basis of size, by their rate of migration under an APPLIED voltage.
Moral & Ethical Concerns:
Ownership - Who should OWN genetic information? This is important due to safeguarding concerns over people’s genetic information. This information is liable to abuse if not carefully concealed.
1. If a person is identified as having a particular DNA seqence for a certain disease then this can be used against them in claiming fair insurance.
2. If a DNA sequence suggests particular ancestry, this should not be used for discrimanatory purposes.
3. No company should utilise this information to their advantage by making a profit.
The idetififcation of allele sequences: a patient’s DNA can be scanned for mutated sequences that may be correlated with future health issues. Some people don’t wish to possess this knowledge. However, if a close relative could be imapcted too, it must be clear as to whether or not the relatives have the right to the information or not…
Genetic screening can be useful with genetic counselling. If a family has a history of a genetic defect, family members can consult a genetic counsellor for advice on the risk for themselves, or potentially, their kids.
Embryos made during the process of IVF can be screened for the presence of alleles leading to conditions like CF & huntigton’s. A choice can then be made to only impant the healthy embryo. A legal framework already exists for the use of ‘spare embryos’ for research!
Parents may wish to screen their child to see whether or not they carry sequences that might pre-dispose them to adult disease. A decsion thus remains on whether to tell the child or not. Furthermore, do the parents have the right to know - it suggests the child’s DNA is a property of the parent.
a)
Ownership - Who should OWN genetic information? This is important due to safeguarding concerns over people’s genetic information. This information is liable to abuse if not carefully concealed.
1. If a person is identified as having a particular DNA seqence for a certain disease then this can be used against them in claiming fair insurance.
2. If a DNA sequence suggests particular ancestry, this should not be used for discrimanatory purposes.
3. No company should utilise this information to their advantage by making a profit.
The idetififcation of allele sequences: a patient’s DNA can be scanned for mutated sequences that may be correlated with future health issues. Some people don’t wish to possess this knowledge. However, if a close relative could be imapcted too, it must be clear as to whether or not the relatives have the right to the information or not…
Genetic screening can be useful with genetic counselling. If a family has a history of a genetic defect, family members can consult a genetic counsellor for advice on the risk for themselves, or potentially, their kids.
Embryos made during the process of IVF can be screened for the presence of alleles leading to conditions like CF & huntigton’s. A choice can then be made to only impant the healthy embryo. A legal framework already exists for the use of ‘spare embryos’ for research!
Parents may wish to screen their child to see whether or not they carry sequences that might pre-dispose them to adult disease. A decsion thus remains on whether to tell the child or not. Furthermore, do the parents have the right to know - it suggests the child’s DNA is a property of the parent.
Genetic Fingerprinting:
a) What are the three things that the sample DNA is dissolved into?
b) What are the stages of PCR? What do you end up with?
c) What machine is used to complete PCR?
d) Provide the temperatures at which eahc step is preformed and explain why…
a)
PCR (Polymerase Chain Reaction) is a semi-conservative replication of DNA in a test-tube. It greatly amplifies the DNA (i.e. making multiple copies of it) and it works rapidly. This makes PCR useful when analysing small or degraded samples.
It all starts with the sample DNA being dissolved in a buffer and being MIXED with:
- Taq polymerase - which is a DNA polymerase from the extremophile bacteria Thermus aquaticus, which lives in hot springs and hydrothermal vents. Taq polymerase has an optimum temperature of about 80-98 degrees (DNA polymerase is an enzyme that plays a crucial role in the synthesis of DNA. It catalyses the addition of nucleotides to the growing DNA strand during DNA replication and other processes such as DNA repair and sequencing).
- Nucleotides containing the 4 DNA bases.
- Short single-stranded pieces of DNA about 10 bases long, called PRIMERS. They are complementary to the start of the DNA strand and bind to it, signalling Taq polymerase to START replication.
b) The STAGES:
- Target/Sample DNA is heated to 95 degrees. This seperates the 2 strands of the sample that bind to form the DNA structure. The heat breaks the hydrogen bonds between the two strands.
- The solution is then cooled. Once it’s cool enough for the primers to anneal to the complementary base sequences on each of the single strands of DNA produced.
- The solution is then heated to 70 degrees. Taq polymerase catalyses the synthesis of a complementary strand by adding complementary nucleotides and catalysing the formation of phosphodiester bonds. This part is called the elongation or extension phase.
- For each fragment of a single double-stranded DNA molecule, 2 identical double strands are produced.
- Repeated many times.
After completing the PCR process, you end up with many copies of the target DNA region, and these copies are the new DNA molecules that were synthesized during the PCR reaction.
Limitation of PCR:
a) There are 5 limitations of PCR go through each.
Limitations of PCR:
a) Before PCR was invnted, copies of genes were made by inserting them inot replicating microorganisms - Fun FACT.
- Contamination - any DNA that enters the system by accident can be amplified and therefore ditort the desired results. The contaminating DNA may be air-borne or come from the experimenter or even contaminated reagents! Most, however, result from the recycling of the apparatus used to complete PCR.
- Error Rate of PCR - all DNA polymerase enzymes sometimes insert the WRONG nucleotide containing the wrong base. They usually proofread and correct their errors, however, the enzyme taq polymerase cannot do this. It makes an error about once in every 9000 nucleotides. After 30 cycles of PCR that error rate becomes 1 in every 300 nucleotides, this is because each cycle copies and multiplies the previous errors and as a result the accumilate!!
- DNA Fragment Sizing - PCR is most efficient for making DNA about 1000-3000 base pairs long because of taq enzyme’s errors. If a lower temperature, higher pH and a proofreading polymerase in addiotion to Taq polymerase are used, a length of 40,000 base pairs can be generated. But many genes, including human genes, are much longer than just 40,000!
- Sensitivity to Inhibitors - molecules in the sample of DNA may act as INHIBITORS and PCR is very sensitive to them.
- Limits of amplification (PCR rate of productivity) - at the start of PCR, the number of DNA molecules made increases exponentially. After about 20 cycles, it slows down.
Gel Electrophoresis:
a)
b)
c)
a) Gel electrophoresis is a technique used in Sanger Sequencing. DNA is extracted from the biological material and cut into thousands of fragments of varying lengths using restriction endonucleases. A ‘restriction endonuclease’ is an enzyme who cuts the sugar-phosphate backbone of DNA molecules at certain nucleotides in the sequence.
b) The fragments are seperated by lengthwith gel electrophoresis, on an agarose gel. Agorose gel is a polysaccharide extracted from seaweed. It makes a gel with pores thorugh which molecules can move thorugh…DNA samples are loaded in at one end of the gel. A voltage is then applied across the entire gel. The phosphate groups of the DNA shall move towards the anode (positive end). The smaller the fragment, the more quickly it can move through the gel as opposed to the larger fragments…The DNA is then seperated into seperate strands by flooding the gel with alkali (hydrogen bonds are disrupted). The alkali gets neutralised later. The electrophoresis trough is then covered with a nylon membrane, which touches the gel and picks up the DNA fragments. This is called southern blotting. Radioactive or luminescent probes contain sequqences comlementary to the to the DNA single strands on the nylon membrane (after southern blotting). Any unbound probes are washed off. A film that is sensitive to X-rays or wavelengths emitted by fluorescent light (that comes from the probes attached to the DNA strand) is then applied overnight to the nylon membrane. the film was exposed to the fluorescent light and so it reveals banding. The pattern is a genetic fingerprint.
c) Probes are short pieces of DNA that are labelled with fluorescent markers used to detect the presence of certain base sequences in another pece of DNA comlementary to the probe…
Short Tandem Repeats:
Less than 2% of our DNA codes for proteins, in sections of teh genome called exons. Those exons have in between them base sequences called introns that do NOT code for proteins. Introns can have up to 13 bases that repeat several hundered times. They are called STRs - short tandem repeats.
STRs are different in differnt individuals and this is what makes a genetic fingerprint unique! The number of repeats of an STR is inherited and so can allow for the identification of people and for tracing family relationships.
DNA profiling in society:
a)
b)
a) Genetic fingerprinting has been a major tool in providing justice - it allows for the identity of individuals to be recognised by using gel electrophoresis to determine the DNA base sequencing in STRs.
Situations in which DNA profiling is used:
1. Establishing Paternity - The DNA from the WBC (white blood cells) are used to conduct the DNA profiles. The bands that result from gel electrophoresisin a child’s DNA are compared to the bands of the mother’s. Any bands they share have been inherited by the child from the mother. The remaining bands come from the father. If they don’t match the alleged father, he is not the biological parent of the child.
2. Twins - Monozygotic twins have identical banding patterns in their DNA profile. This isn’t the case with dizygotic twins.
3. Siblings - People who have been adopted may wish to confirm their alleged biological siblings are i fact their blood relatives. Half their genes should be the same. DNA profile can show many similarities.
4. Immigration - Proving your relations/relatedness for visa applications
5. Forensic studies - criminal cases & finding out the identity of the individual who commited the crime.
b) Pros of DNA profiling:
- Doesn’t require an invasive method to obtain biological sample - mouth swabs, urine and hair sample are all aceptable methods of obtaining an individual’s DNA as opposed to blood samples.
- The technique can be used on samples that would be too small for blood testing.
- DNA profiling can rule out non-matches of DNA samples, to exonerate (free someone from blame) who have wrongly been accused of a crime.
Cons of DNA profiling:
1. Some people condsider that any request of their DNA is a violation of their rights to privacy.
2. DNA profiles are held in computer databases which means that they’re prone to being hacked. Individuasl will then suffer a loss of their privacy.
3. Profiles are’nt absolute. There is a 1 in a million chance that the outocome dtermined by DNA profiling is incorrect. To reduce uncertainty more sample can be used and tested.
4. Safeguarding of the database is key and access is vital for regulation of information. If mis-used health insurers for example can deny covergae claims. Prospective employers can deny/discriminate against individuals.
5. Wrongful convictions may result if not carried out correctly, people undertaking the testing are untrustworthy and can influence the results of the tests or if DNA evidence is planted at the crime scene.
c) Remember the difference between DNA profiling and DNA sequencing. DNA profiling is all about curating the details of a sequence of the short tandem repeats of an individiual! The STRs. Conversely, DNA sequencing is all about obtianing the genetic sequence of a DNA sample, i.e. gaining information about the nucleotide sequence.
Genetic Engineering:
a) Genetic engineering allows genes to be manipulated, altered and transferred from one organism to another organisms making a genetically modified organism labelled GM organism. Applications of genetic engineering include the transfer of genes or gene fragments into:
-Bacteria, so they make useful products, such as insulin.
-Plants and animals, so that they aquire new characteristics, e.g. resistance to certain diseases
-Humans to reduce the effects of genetic diseases such as DMD.
When genetic material from 2 species is COMBINED, the result is a recombinant DNA (this means a DNA that is results from the combination of 2 different species). Therefore, we can also call genetic engineering, recombinant DNA technology
