Biotechnology - Chapter 10 Flashcards
Polymerase chain reaction
a technique used in molecular biology for producing multiple copies of DNA from a sample; used in DNA fingerprinting and identifying disease.
3 Steps to PCR testing
- denaturing
- annealing
- extension
Denaturing
- heat is used to seperate two strands of DNA
- temperatures of 94-96ºC are used to break hydrogen bonds holding the two strands together
Annealing
- during this process, temperatures decrease to 50-60ºC
- this allows short strands of DNA (primers) tp bind to the single DNA strands
Extension
- also known as elongation mimics DNA replication
- this process is carried out at 68-72ºC
- DNA polymerase is used to join new, complimentary nucleotides to sections originating with primers
- the nucleotide chain extends and creates a new strand of DNA
- this new strand is not as long as the original strand as it starts at a primer
- DNA polymerase attaches to the double stranded DNA
- Primers initiate DNA replication
Taq Polymerase
heat sustainable DNA polymerase
- does not denature when heated
- optimal temp 68-72ºC
Gel electrophoresis
a process used to seperate charged molecules based on their size by pushing them through gel.
Steps to gel electrophoresis
- DNA pieces placed in wells in semi-solid gel that is immersed in a solution of electrolyte
- (electrodes at either end of gel) negative electrode is closest to the DNA and positive is on the opposite side
- when electric current is passed through the gel, negatively charged DNA moves towards the positive electrode. when current is stopped, smaller DNA is further from negative electrode as they move faster.
- results in a pattern of bands, which is a persons DNA profile/DNA fingerprint
Micropipette
a fine pipette used to measure and transfer very small volumes of liquid.
- has disposable tips that can be put on and off the end of the pipette without contact, reducing any chance of cross-contamination.
DNA ladders
- run at the same time as samples and has known lengths
- used to compare lengths of DNA samples
Visualising DNA
ethidium bromide, methylene blue, DNA probes
Ethidium bromide
can be added to agar. as DNA moves through, it picks up some of the chemical. special ultraviolate light is shown over the gel and DNA fluoresce
Methylene blue
a dye that binds to DNA. when gel is soaked in dye, DNA stains a deeper blue - visible to the naked eye.
DNA probes
are short sections of a single strand of DNA with a radioactive fluorescent molecule that binds to DNA.
DNA sequencing
the determination of the precise order of nucleotides in a sample of DNA.
Dideoxyribonucleotide
a modified deoxyribonucleotide that lacks a hydroxyl group of the sugar component.
Sangers method
- adds dideoxyribonucleotide to the growing strand
- this synthetic nucleotide stops the elongation as there is no OH group for the next nucleotide to attach to
- this happens to each nucleotide, creating different lengths of DNA
- can be separated using gel electrophoresis - knowing which base was added to create each length allows determination of order of nucleotides
Uses of DNA sequencing
- identifying mutations
- identifying sickle-cell anaemia, cystic fibrosis and other forms of cancer
- for maternity and paternity tests
- to compare species
Ethical considerations with genetic info
autonomy - the right to be self determining and choose whether or not to be testes on
confidentiality - the use of genetic info is treated sensitively, and is accessed only by those who are authorised to access it.
equity - right to fair and equal treatment, regardless of genetic info
privacy - right to be left alone and make decisions independent of others.
Genome
a complete set of genetic material in a cell; an organisms complete set of DNA
Comparative genomics
the comparison of genome sequences of different species.
- allows researchers to find areas of similarity and differences
- provides effective means of studying evolutionary changes
- humans share 98% of DNA with chimpanzees
Junk DNA
non-coding sequences that have no apparent function and appear to serve no purpose.
- more closely related species have more junk sequences in common.
Endogenous retrovirus
non-functional DNA (viral sequence). they store their genetic information as RNA.
Reverse transcription
upon entering a cell, a retrovirus copies its RNA genome into DNA in a process known as reverse transcription. the DNA then becomes inserted into one of the host cells chromosomes.
When is a retrovirus endogenous?
only becomes endogenous if it is inserted into a cell who’s chromosome will be inherited by the next generation - an ovum or sperm cell.
- offspring of the individual will then have a copy of the ERV in the same place, in the same chromosome, in every single one of their cells.
Mitochondrial DNA
DNA found in the mitochondria of the cells, rather than the nucleus.
- in the form of small circular molecules
- 5-10 of these molecules in each mitochondrion
- has 37 genes
37 Genes of the mitochondria
24 code for making transfer RNA molecules, which are involved in protein synthesis.
13 have instructions for making some of the enzymes necessary for the reactions of cellular respiration.
Inheritance of mtDNA
