Crucial

Question 1

what are the stages in apoptosis

Answer 1

• Hydrolytic enzymes break down the proteins in the cell cytoskeleton
• Cell shrinks, cytoplasm condenses, organelles become tightly packed
• Apoptotic blebs form as the CSM shape changes
• Nuclear envelope degrades, chromatin condenses and DNA fragments
• Cell breaks into apoptotic bodies (membrane-bound vesicles) that are engulfed by phagocytes
• No cell debris in the extracellular fluid, surrounding cells not damaged

Question 2

what are the functions of apoptosis

Answer 2

1. To model the body during development eg. digits, metamorphosis the cells undergoing apoptosis release chemical signals which stimulate mitosis
2. Destroy harmful T lymphocytes during development of immune system, preventing harm to body cells
3. Cells in senescence (50+ mitotic divisions)
4. Cells experiencing cell stress eg. irreparable genetic damage

Balance between apoptosis + mitosis regulated by hormones, transcription factors, cytokines, nitric oxide

Question 3

creating a genetic fingerprint

Answer 3

Amplify by PCR: polymerase chain reaction. DNA to be amplified denatured at 94’C- H bonds break- single stranded DNA. Cool to 55’C to anneal primers CBP to regions flanking required gene in the sample DNA. Extension, Taq polymerase(thermostable DNA polymerase), 72’C, builds two new polynucleotides. 2 identical DNA strands

DNA is cut/cleaved within the STRs/microsatellites into fragments using restriction endonucleases.

Gel electrophoresis; separates DNA fragments by migrating through agarose gel based on size- electric current- negatively charged due to phosphates. Smallest moves furthest, largest move slowest.

Southern blotting/hybridisation; radioactively labelled DNA probe, i.e. short DNA sequence, labelled + introduced to detect complementary DNA by hybridisation. Transferred onto permanent nylon/nitrocellulose blot by heating to denature DNA, probe hybridises with SS DNA, blot is washed of probe, autoradiographed to detect radioactivity. Placed on X-ray film. Development reveals dark bands.

Question 4

key points in Chain Termination Sequencing

Answer 4

DNA cut into manageable fragments, separated, mixed with primer, DNA polymerase, excess of normal nucleotides, terminator nucleotides with coloured fluorescent tags.

Thermal cycler

Terminator base stops synthesis of DNA

DNA fragments separated by length through minute capillary tube

Fluorescent markers used to identify final base, lasers detect colours to find complementary sequence

Question 5

explain High Throughput Sequencing

Answer 5

• uses chain synthesis au lieu de termination
• DNA fragments are 300-800 bases
• denatured to SS & immobilised
• primer + DNA polymerase added + enzymes that catalyse a reaction that produces light when a nucleotide is added to chain
• only one nucleotide added at a time; if light emitted, this is the nucleotide in the sequence

Question 6

what is Next Generation Sequencing

Answer 6

eg. nanopore sequencing

Two proteins create a nanopore; the upper protein unzips/unwinds the DNA, lower protein creates a pore through Plipid bilayer

Flow of ions through pore creates a current, each base alters current flow in a different way + current is determined as each base passes through pore

Question 7

what is genetic engineering

Answer 7

the addition of an extra segment of DNA

using a vector- plasmid/virus

A plasmid is very small segment of circular DNA with an origin of replication. Present naturally in bacteria.

Question 8

how to gobtain the gene of interest

Answer 8

1. OBTAIN GENE OF INTEREST 1 : DNA probes from southern blots of gel e. to find position of gene, which is isolated + amplified.
2. OBTAIN GENE OF INTEREST 2 : obtain gene form respective mRNA eg. ß-cells produce insulin + have mRNA for insulin in large quantities. Reverse transcriptase MAKES DNA FROM THE RNA. Obtained from retro-viruses. cDNA inserted into plasmid, inserted into bacterial cell. Bacterial cell grown in fermenter, gene cloned. Protein expressed, extracted.

Question 9

how to isolate a plasmid

Answer 9

Bacterial cells lysed with detergent + centrifuged. Bacterial DNA is in pellet, plasmid DNA in supernatant.

Question 10

How to splice the DNA of interest into the plasmid

Answer 10

Restriction endonucleases cut at specific base sequences. RECOGNITION SITES.

• sticky ends or blunt ends.
• used to cut the DNA of interest + plasmid DNA
• sticky ends of DNA of interest & plasmid DNA H bond together and DNA ligase used to join sugar-phosphate backbone.

Forms recombinant DNA/ hybrid DNA/ hybrid plasmid, which is reintroduced into the host cell by transformation. often by electroporation.

Other hosts = yeast (eu), plant eg. pesticide resistance, animal eg. pharming

Produces transgenic/ GM organism.

Question 11

how to count colonies

Answer 11

• viable plate count*
• serial dilution*

Question 12

parts of the brain

Answer 12

Cerebrum- cognitive factors eg. sexual arousal

Hypothalamus- sends impulses

Medulla oblongata-

Chemoreceptors + baroreceptors in carotid arteries, aorta