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.
Semi-conservative nature of DNA replication (COMPLIMENTARY BASE pairing when forming new DNA helixes)
- The DNA base sequence of the parent strands act as template for the new strands.
- Complementary bases form hydrogen bonds –stabilizing the structure. This would not happen if the bases were not complementary to each other.
- Adenine pairs only with Thymine. Cytosine pairs only with Guanine
- The rule of joining matching nucleotides is called complementary base pairing. This is important to maintain & conserve the correct sequence of bases in the molecule and to create an identical copy, but it is also essential for protein synthesis.
- Complementary base pairing ensures a high degree of accuracy when strands are assembles.
3 different ideas of how DNA might be replicated, existed at that time: semi-conservative, conservative (photocopier), dispersive (chunk itself into nucleotide segments to be copied)
Later experiments (Meselson-Stahl experiment) proved that DNA replication is 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.
OSSD -RECOMBINANT DNA & RESTRICTION ENZYMES
- what is recombinant DNA?
RECOMBINANT DNA
a fragment of DNA composed of sequences originating from at least two different sources.
OSSD
- what are RESTRICTION ENZYMES/endonucleases?
RESTRICTION ENDONUCLEASES.
otherwise known as restriction enzymes.
they recognize a specific short sequence of nucleotides (the target sequence) on a strand of DNA and cut the strand at a particular point within that sequence. This point is known as the restriction site.
Recognize palindromic base sequences in DNA
GAATTC
CTTAAG
Cut DNA (both strands) at this point Restriction endonucleases animation
Different R.E. for different sequences
Usually 4 to 12 base pairs
Hundreds have been identified and isolated
A particular enzyme usually makes numerous cuts
The cut pieces of DNA are called “restriction fragments”
Most restriction endonucleases produce a staggered cut that leaves a few unpaired nucleotides remaining on a single strand at each end of the restriction fragment.
these short sequences are often referred to as sticky ends
they can form base pairs with other short strands having complementary sequences.
OSSD
- The genetic code is universal, this means that base sequences can be transferred from one organism to another without changing their function
-Gene transfer
In gene transfer techniques, the gene for a favourable trait is identified on one organism and transferred to another
Gene transfer can be used in:
Gene therapy: to repair a faulty gene in an organism
Industry/Medicine: to produce large volumes of a desired protein
Industry/Agriculture: to genetically modify organisms to favourable characteristics
Plasmids
- Cheap machine (bacteria) are used to duplicate genes and the proteins they code for
Small loop of DNA independent of chromosome
Double-stranded
Replicates independently
Can be 100s of one type per cell
Used to make multiple copies of genes-Cloning vectors
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 (DAE)
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
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.
