D1.1 DNA Replication Flashcards
Outline what is meant by DNA Replication
DNA replication is the production of exact copies of DNA with identical base sequences. It takes place in the S phase of interphase and before cell division, before mitosis and meiosis and before binary fission in prokaryotes.
Explain how electrophoresis works
Gel electrophoresis is a process that is used to separate molecules such that proteins and fragments of nucleic acids. Electrophoresis is central to investigating sequence of bases in particular length of DNA, known as DNA sequencing. It is also used in the identification of individual organisms and species, known as genetic profiling.
**How it works:
Proteins and nucleic acids are separated based on overall charge or mass. Separation is due to factorsDifferential migration of these molecules through a supporting medium- either agorose gel or polyacrylamide gel (PAG). In these media, the tiny pores in the gel act as a moelcular sieve. Small particles are able to move quickly, but larger molecules move slower.
The electric charge that molecules carry- it is the phosphate groups in DNA fragments that give them a net negative charge. Consequently, when these molecules are placed in an electric field, they migrate towards the positive pole.
DNA fragments are produced b the actions of one or more restriction enzymes. Different restriction enzymes cut at particualar base sequences, as and where they cut along the length of DNA. Consequently, fragments of different lengths are produced.
A series of groves or wells are cut close to one end of the gel, which is then submersed in a salt solution that conducts electricity. Then a small quantity of a mixture that will be separated is placed in a well. Several different mixtures can be separated in a single gel at one time.
After separation, the fragments are not immediately visible, they are tiny and transparent. They are either identified by gene probes or DNA stains. A gene probe is a single stranded DNA with a base sequence that is complementary to that of a particualr fragment or gene who we want to find. The probe must be made radioactive such that when treated gel is exposed to X ray film, the presence of the fluorescent stain is attached. It will then fluoresce in UV light, hence indicating the presence of the particular fragment or gene that we want.
Stains are used to immediately locate the position of all DNA fragments once applied. Stains include methylene blue which stain gel and DNA, but is less sensitive and colour fades quickly. Another tain is ethidium bromide.
Outline the role of the buffer and power supply in electrophoresis
Explain how helicase and DNA polymerase work in DNA replication.
Firstly, DNA replication is the disruption of the hydrogen bonds that hold the two strands of double-stranded DNA together, and the DNA is unzipped. Enzyme helicase unwinds the DNA, for DNA polymerase to later act on. As helicase unwinds the DNA double helix at one region, breaking the hydrogen bonds that hold strands together and then temporarily keep the strands of the helix separated. Then, unpaired nucleotides are exposed and surrounded by a pool of free-floating nucleotides.
Secondly, both DNA strands act as template strands for replication. Complementary nucleotides line up opposite each base, such as adenine with thymine, cytosine with guanine. Hydrogen bonds form between these complementary base pairs and hold them in place.
Finally, a condensation reaction links together the sugar and phosphate groups of adjacent nucleotides to form new strands. The reaction is catalysed by DNA polymerase. DNA polymerase also corrects any mistakes made during eplication such as bases wrongly pairing with one another. As a result, two strands are formed identical to the original strands.
Describe the applications for PCR and gel electrophoresis.
Gel electrophoresis is the process used to separate fragments of DNA or proteins, on a polymer gel, according to size and overall change. It is applicable in DNA sequencing and genetic profiling. In electrophoresis, proteins or nucleic acid fragments are separated on the basis of their overall charge and mass.
Describe 2 limitations of PCR
Firstly, knowledge of the DNA or amino acid sequence of a desired gene is needed to synthesize flanking nucleotide primers, which is one limitation.
Secondly, non-target DNA sequence may be amplified instead of the desired sequence, as primers are short nucleotide sequences and may not be specific enough.
Note*:DNA primers are short sequences of single-stranded DNA which are made synthetically with base sequences complementary to one end of the DNA.
What are the factors which influence gel electrophoresis?
Gel electrophoresis proteins or nucleic acid fragments are separated based on their overall charge and mass. Separation is due to the following factors:
1) Differential migration of these molecules through a supporting medium- either agorose gel or polyacrylamide gel (PAG). In these media, the tiny pores in the gel act as a moelcular sieve. Small particles are able to move quickly, but larger molecules move slower.
2) The electric charge that molecules carry- it is the phosphate groups in DNA fragments that give them a net negative charge. Consequently, when these molecules are placed in an electric field, they migrate towards the positive pole.
Hence, this is the double principle of electrophoretic separation: Separation occurs on the basis of size and charge.
Describe the roles of helicase and DNA polymerase in DNA replication
Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds.
DNA polymerase links nucleotide together to form a new strand, using the pre-existing strand as a template.
State the biological term for random changes in the nucleotide sequence.
Mutations.
Define the term “DNA primer”
A DNA primer is a short sequence of single-stranded DNA used in PCR and is made synthetically with a base sequence complimentary to one end of the DNA.
Define genetic profiling
Genetic profiling is the identification of individual organisms or species using DNA.
What is the function of variable number tandem repeats (VNTR)
VNTRs are short base sequences that show variation between individuals in terms of number of repeats. These major lengths of non-coding DNA are used in genetic profiling.
What are some applications of genetic fingerprinting
Indentification of suspects. Samples are taken from the crime scenes. DNA from both victims and suspects, as well as from others who have certainly not been involved in the crime used as a control sample. All these samples are profiled. Great care is taken such that cross-contamination does not occur.
Identification of corpses. Bodies that are too decomposed for recognition may be identified, as might parts of the body remaining after violent accudents or natural disasters. DNA samples from body tissues are taken and their profile compared with those of close relatives or with DNA obtained from cells recovered from personal effects , where they are available.
Determining paternity. A range of samples of DNA are analysed side by side, of the people who are possibly related. The banding pattern are then compared, and because a child inherits half of their DNA from the mother and half from the father, the bands in a child’s DNA fingerprint that do not match those of its mother must come from the child’s father.
Explain some applications of PCR and gel electrophoresis?
Genetic profiling. The majority of DNA is not composed of genes but non-gene regions which include repeating short base sequences. Many are joined together in large clusters. These are used to profile, as each person has a unique amount of these sequences which are properly known as Variable Number Tandem Repeats. Numbers are distinct, half from our mother half from our father, consequently everyone has a unique sequence of nucleotides in DNA except identical twins who share the same pattern. To produce a genetic fingerprint or profile, a sampe of DNA is cut with retsiction enzyme close to VNTR regions. Electrophoresis is then used to eparate the fragments according to length and size. and the result is a pattern of bands.
Outline what is meant by semi-conservative replication.
