6.3 MANIPULATING GENOMES Flashcards
Define genome
- The complete set of genes in a cell
Define sequencing
- Identifying the locus and the order of DNA bases for particular genes
Describe what genome sequencing gives information about
- The location of genes
- Evidence for evolutionary links between organisms
Define locus
- The location of a gene on a region of chromosome
State the three types of sequencing
1) Sanger sequencing
2) Automated sequencing
3) High throughput sequencing
Compare the three types of sequencing
- Sanger sequencing is slow but high accuracy and requires manual reading of results
- Automated sequencing uses sanger techniques so slow but accurate however computer reads results
- High throughput allows for whole genome sequencing and is very fast but less accurate and computer reads results
Explain why during sequencing:
-Copies of a gene are broken into random fragments
- Fragments are sequenced separately
- Sequences are put back together in correct order by matching overlapping fragments
- Because whole genes and genomes are too large to be sequenced in one fragment
State three uses of genome sequencing
- Identify phylogenetic relationships
- Predict primary structure of proteins from genes by predicting the sequence of amino acids in polypeptides
- Create gene probes for disease screening in synthetic biology
State what gene probes are
- Radioactive/fluorescent fragments of DNA that are complementary to known target sequences of DNA
- (Are easily identifiable)
State what screening programmes do
- Search for specific disease alleles within genomes
State where sequences genes are stored
- Gene banks
Define bioinformatics
- the digital storage of large quantities of biological data
State four features of bioinformatics
- Sequences for existing healthy or disease alleles are stored
- Allows for rapid comparison between newly discovered sequences and stored ones
- Existing protien primary structure stored
- Can model possible 3D shapes of new proteins coded for the the newly discovered alleles
Define tandem repeats
- Repetitive segments of DNA that do not code for proteins
Define DNA profiling
- Forensic technique that analyses the non-coding tandem repeat DNA to compare DNA samples
Describe the general procedure of DNA profiling for forensics
1) DNA is obtained from the crimescene (through sperm/saliva/blood/bone)
2) Restriction enzymes then digest the DNA which cut the DNA at specific recognition sites forming fragements
3) These fragements are separated by gel electrophoresis and are stained
4) A banding pattern can be seen
5) The suspects DNA is treated with the same resitriction enzyme and subjected to electrophoresis
6) The banding patterns of both DNA are compared to see if theres a match
Define banding patterns on DNA
- They are patterns of light and dark transverse bands on a chromosome
State the three uses of DNA profiling
1) Forensic science
2) Reveal maternity/paternity
3) Analysis of huntingtons disease/diseases
Define polymerase chain reaction (PCR)
- Where a short length of DNA is rapidly and synthetically amplified into millions of copies
- (Artificial DNA replication)
State what four factors polymerase chain reaction (PCR) relies on about DNA molecules
1) DNA is made of two antiparrallel backbone strands
2) Each strand has a 5’ end and a 3’ end
3) DNA grows from the 3’ end only
4) Base pairs have complementary base pairing
State three ways in which polymerase chain reaction differs from DNA replication
1) Only short sequences of DNA can be replicated, not entire chromosomes
2) It requires primer molecules to make the process start
3) It requires cycles of heating and cooling for : strand separation, binding primer to the strands and for the DNA to be replicated
Explain why a primer is needed for polymerase chain rection (PCR) but not for DNA replication
- PCR consists of using single stranded DNA
- DNA polymerase is not able to bind to single stranded DNA on its own, thus PCR requires a primer
Describe in detail the steps of polymerase chain reaction (PCR)
1) A sample of DNA is mixed with DNA nucleotides, primers, Mg²⁺ and Taq DNA polymerase
2) The mixture is heated to 95°C to break hydrogen bonds between complementary nucleotide base pairs and thus denature the double stranded DNA into two single strands
3) The mixture is cooled to 68°C so the primer can bind by hydrogen bonding to one end on each single strand of DNA
4) This results in a small section of double stranded DNA at the end of each single strand
5) Taq DNA polymerase can now bind to this double stranded region
6) The temperature is raised to 72°C as its optimum temp for taq DNA polymerase enzyme and to keep DNA single-stranded
7) The tag DNA polymerase then catalyses the addition of complementary DNA nucleotides to the single stranded molecule, starting at the primer end and proceeding in the 5’ to 3’ direction
8) Once tag DNA polymerase has reached the other end of the DNA molecule, a new double stranded DNA forms
Describe three uses of polymerase chain reaction (PCR)
1) Tissue typing (the donor and patient tissues can be checked before transplant to reduce the risk of rejection)
2) Detection of mutations (sample of DNA is sampled for presence of a mutation that can lead to a genetic disease)
3) Forsenic science (to identify criminals or parentage)
Define electrophoresis
- Process that separates DNA fragements (nucleic acid fragments) or protein molecules of different sizes
Describe the process of electrophoresis
1) DNA sample first digested with restriction enzymes to cut them into fragments at specific regions
2) DNA sample placed in a well at the negative electrode end
2) Current is applied for a fixed time
3) DNA fragements migrate from the negative end to the positive end
4) Larger fragments move more slowly than smaller fragments, so smaller fragemnts travel further
5) Radioactive or fluorescent probes are added
6) (If radioactive - nylon blotting/autoradiography used to locate fragments) (if fluorescent - uv light used to locate fragments)
Why does DNA have an overall negative charge
- Due to its many negative phosphate groups
State how proteins are separated via electrophoresis
- Using the charged detergent (SDS) in electrophoresis
Explain the effect of SDS in protein electrophoresis
- SDS added equalises the surface charge on the molecules
- This allows proteins to separate as they move through the gel, according to their molecular mass
State the condition that can be tested for by SDS electrophoresis
- Sickle cell anaemia
State three uses of probes
1) To locate a specific gene needed for use in genetic engineering
2) To identify the same gene in a variety of different genomes from different species when genome comparing
3) To identify presence or absence of a specific allele for a particular genetic disease
Define microarray
- A fixed surface with many different probes
- DNA is applied onto it to reveal mutated alleles that are complementary to the fixed probes
State the gel used in electrophoresis
- Agarose gel
Define DNA ligase
- The enzyme that catalyses the joining of sugar and phosphate groups within DNA
State the steps of **genetic engineering
1) The required gene is obtained
