sl DNA replication Flashcards
DNA replication as production of exact copies of DNA with identical base sequences
- Students should appreciate that DNA replication is required for reproduction and for growth and tissue replacement in multicellular organisms.
- Each molecule formed by replication consists of one new strand and one old strand conserved from the parent DNA molecule. The result of replication is therefore is two DNA molecules, both composed of on original strand and a newly synthesized strand.
- DNA Replication is required for organism to grow, reproduce and repair
Reproduction
Offspring need copies of the base sequences of their parents, so parents must replicate their DNA when reproducing sexually or asexually.
Growth
Multicellular organisms increase their size by increasing their number of cells (individual cell size cannot grow due to SA : V ratio).
Tissue replacement & repair
Tissue replacement and repair in multicellular organisms requires cell division in tissues where they have been lost or damaged (e.g. skin cells which have been worn away, wounds…). New cells with a full set of an organism’s base sequences are needed for that, so DNA must be replicated.
Semi-conservative nature of DNA replication and role of complementary base pairing
- Students should understand how these processes allow a high degree of accuracy in copying base sequences.
- The two strands of DNA come apart, and each strand acts as a template for the DNA replication.
- The new strands are formed by adding nucleotides one by one and linking them together.
- When replication is complete, there are two DNA molecules, both composed of an original strand and a newly synthesized strand.
- For this reason, DNA replication is referred to as semi-conservative.
Role of helicase and DNA polymerase in DNA replication
- Limit to the role of helicase in unwinding and breaking hydrogen bonds between DNA strands and the general role of DNA polymerase.
- The enzyme helicase unwinds the DNA double helix and separates the two strands by breaking hydrogen bonds. Energy (ATP) is required for this process
- The enzyme DNA polymerase adds free nucleotides one by one in positions on the template strand where hydrogen bonds between complementary bases can form. Once in the correct position DNA polymerase forms a covalent bond between the sugar of the previous and phosphate group of the new nucleotide.
Polymerase chain reaction and gel electrophoresis as tools for amplifying and separating DNA
- Students should understand the use of primers, temperature changes and Taq polymerase in the polymerase chain reaction (PCR) and the basis of separation of DNA fragments in gel electrophoresis.
- The polymerase chain reaction (PCR) is an automated procedure used to to copy specific sections of DNA molecules in a cell sample (e.g. blood, tissue cells, semen…). PCR machines follow repeated cycles of heating and cooling to replicate a small quantity of DNA using a special type of polymerase (Taq).
- Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230)
- Once large quantities of DNA are created, other laboratory techniques are used to isolate and manipulate the sequences.
Polymerase chain reaction as tools for amplifying and separating DNA
- Students should understand the use of primers, temperature changes and Taq polymerase in the polymerase chain reaction (PCR) and the basis of separation of DNA fragments in gel electrophoresis.
- Thermus aquaticus as an extremophile (archea) bacteria living in especially harsh conditions such as hot springs. It too, needs to replicate using DNA polymerase – therefore it must be heat resistant. This useful for its purpose in PCR.
- Thermus aquaticus’ polymerase is often abbreviated to “Taq”. and is frequently used in polymerase chain reaction (PCR) because the enzyme withstands the high temperatures required in this process without being destroyed (denatured).
PCR Process (DRE)
repeats every 20-40 cycles
PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:
- Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min)
- Annealing – DNA primers, which are short RNA sequences serving as starting points for polymerase, attach to the 3’ ends of the target sequence ( 55ºC for 1 min)
- Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min) by adding nucleotides.
Gel electrophoresis as tools for amplifying and separating DNA
- Students should understand the basis of separation of DNA fragments in gel electrophoresis.
GET BOOKLET - stepsss
Sometimes it is necessary to separate DNA molecules by length. This is done using a process called gel electrophoresis.
The gel used acts like a molecular sieve – it allows small molecules to pass further through than long ones. Prior to separating the DNA it must be cut into little pieces.
- Obtain DNA sample + amplify in PCR machine
- Restrictive enzymes
- DNA samples in wells on gel
- DNA is negatively charged, so…
- The movement of molecules/distance travelled depends on SIZE/LENGTH
Applications of polymerase chain reaction and gel electrophoresis
- Students should appreciate the broad range of applications, including DNA profiling for paternity and
forensic investigations.
- Both techniques, PCR and gel electrophoresis, are required to establish a DNA profile or fingerprint.
- DNA profiling is the process of comparing short DNA sequences of different indivuduals and look for matches in fragement length. The main applications are in crime scene/forensic investigations and paternity investigations.
- The technique of PCR combined with gel electrophoresis has many applications:
Crime investigations
Paternity testing
Testing for coronaviruses
apps continued
- DNA Profiling requires the use of PCR and gel electrophoresis, as well as specific sections on the DNA which do not code for a protein. These sections are non-coding DNA and are referred to as STRs and VNTRs.
- Out of all the DNA, only ca. 1.5-3% of the genome is coding DNA - these are shorter sections of bases of DNA that have a unique sequence.
- Highly repetitive sequences, such as (short) tandem repeats are used for DNA profiling.
apps - what are STRs and VNTRs?
- STRs and VNTRs are short repetitive sequences on the DNA which differ slightly between individuals and therefore allow a specific DNA fingerprint to be produced for every person.
- Variable Number Tandem Repeats, VNTR (minisatellites) have longer base sequences (hundreds of base pairs) repeated few times
- Short Tandem Repeats, STR (microsatellites) have shorter base sequences (2-6 base pairs) repeated many times.
Making a DNA Profile
A DNA sample is collected (from blood, semen, saliva), extracted and amplified using PCR (to increase the amount).
Satellite DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments.
DNA fragments are separated according to length of fragment and therefore number of repeats using gel electrophoresis.
Fragment length will differ between individuals due to the variable length of their short tandem repeats, as will the number of fragments.
A pattern of bands of DNA is produced on the gel that is always the same with DNA taken from one individual. This is the individual’s DNA profile. It can now be compared with other samples and the DNA fragment length can be estimated using a DNA ladder.
what’s a DNA ladder?
A DNA ladder is a marker which contains DNA fragments of exactly known sizes. The marker is usually applied in the first lane and allows an estimation of the fragment sizes of unknown samples on the gel.
