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Biology Alevel > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

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1
Q

What is Sanger sequencing ? (Chain termination method)

A

Uses modified nucleotides called DIDEOXYNUCLEOTIDES
- they pair with the complementary nucleotides on template strand during DNA replication
- when DNA polymerase encounters dideoxynucleotide on developing strand it stops replicating - hence CHAIN TERMINATING metho

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2
Q

Chain termination method ?

A
  1. 4 test tubes that contain the singe strand template DNA to be sequenced, DNA polymerase, DNA primers, free nucleotides A, C, T, and G, and 1/4 types of dideoxynucleotide; either A, C, T, or G
  2. Test tubes incubated at temp that allows DNA polymerase to function
  3. Primer anneals to the start of single stranded template - produce short section of double stranded DNA at start of sequence
  4. DNA polymerase attaches to double stranded section/begins DNA replication using free nucleotides in test tube
    H bonds form between complementary bases on nucleotides
  5. DNA polymerase can insert one of the dideoxynucleotides by chance -> termination of DNA replication
  6. Complementary DNA chains produced vary in length , as when the dideoxynucleotides are inserted varies
  7. Once incubation period is over, the new complementary DNA chains separated from TEMPLATE DNA
  8. Single stranded DNA separated according to length using GEL ELECTROPHORESIS - smaller fragments travel further
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3
Q

What is high throughput sequencing ?

A

Describes multiple DNA sequencing technologies that allow simultaneous sequencing of multiple DNA strands
- faster /cheaper way to sequence genomes
Most methods are now AUTOMATED instead of manual interpretation - faster

E.g : capillary gel electrophoresis
Nanopore sequencing /pyrosequencing - next generation sequencing methods (newer)

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4
Q

What are high throughput sequencing methods useful for?

A

Fields fo study in:
Evolutionary biology /classfiication
Personalised med
Disease diagnosis

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5
Q

What is DNA Sequencing ?

A

Technique that allows genes to be isolated +read

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6
Q

What is a genome?

A

The complete set of genetic material present in a cell/organism

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7
Q

What is bioinformatics ? How has it allowed for comparison between genomes ?

A

Field of biology that involves the storage, retrieval, and analysis of data from biological studies
- studies may generate data on DNA /RNA /proetin sequences/relationship between genotype and phenotype
- large databases contain info about organisms gene sequences/ amino acid and protein sequences
- when genome is sequenced , bioinformatics allows scientists to make comparisons with the genomes of other organisms using the databases
- see how closely related organisms are

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8
Q

How is genetic variation and evolutionary relationships investigated by comparison of genomes?

A

Species with high level of genetic variation will have larger no. Differences in base sequences between individuals
Species with small no. Differences between their genomes likely to share more recent common ancestor than species with large no. Differences
- cytochrome C proetin can be used for comparison

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9
Q

How can genome sequencing aid the understanding of genotype-phenotype relationships?

A

Explored by stopping the expression of different genes /observing its effect on the phenotype
- when organisms genome sequence is known , scientist can target specific base sequences to ‘knock out’

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10
Q

How can genome sequencing help in epidemiology?

A

Genomes of pathogens sequenced /analysed to aid research and disease control
- highly infectious strains can be identified
- The ability of a pathogen to infect multiple species can be investigated
- most appropriate control measures can be implemented
- Potential antigens for vaccine production can be identified

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11
Q

How has gene sequencing led to development o synthetic biology?

A

genetic code used to predict the amino acid sequence within a protein
- can then predict how the new protein will fold into its tertiary structure
This information can be used in synthetic biology

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12
Q

What was the human genome project?

A

Began in 1990 - international,collaborative research programme
—> publicly funded so no commercial interests/influence

DNA samples taken from multiple people around the world, sequenced, and used to create a reference genome
Labs around the globe sequenced different sections of specific chromosomes

By 2003 the human genome sequenced to 99.9%accuracy
- finished genome was over 3 billion base pairs long but only about 25,000 genes (quite low)

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13
Q

Applications of the human genome project ?

A
  • info from project used to tackle HUMAN HEALTH ISSUES - nd goal of finding CURES
  • Scientists found a correlation between changes in specific genes and the likelihood of developing certain inherited diseases (cancers, Alzheimer’s )
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14
Q

What is a proteome and why is it difficult to determine the proteome for humans?

A

Proteome: full range of proteins produced by the genome
Large amounts of NON CODING DNA present in genomes

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15
Q

Why is the proteome larger than the genome?

A

Alternative splicing - allows for single gene to produce multiple proteins
Post transitional modification of proteins

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16
Q

What is synthetic biology? What does it involve?

A

recent area of research that aims to create new biological parts, devices, and systems, or to redesign systems that already exist in nature

Involves : large alterations to organism’s genome
New genome can be made using existing DNA sequences or new sequences
- new sequences designed/written so they produce specific proteins

17
Q

Well known use of synthetic biology?

A

Production of ARTEMISININ - antimalaral drug

Scientists constructed a DNA sequence for a new metabolic pathway containing genes from BACTERIA, YEAST and A.annua- plant
- pathway results in production of ARTEMISINIC acid - precursor to artemisinin

Pathway can be inserted into yeast cells which will produce artemisinic acid
This precursor is converted to ARTEMISININ by inexpensive process

18
Q

What is polymerase chain reaction (PCR) for?

A

In vitro method of DNA amplification
- to produce large quantities of specific fragments of DNA or RNA from very small quantities - for analysis

19
Q

What does each PCR reaction contain?

A

Target DNA/RNA to be amplified
Primers (forward/ reverse)- short sequences of single-stranded DNA* complementary to the 3’ end of the DNA or RNA being copied.
DNA polymerase - enzyme used to build new DNA/RNA strand (normally TAQ POLYMERASE)
Free nucleotides - used in construction of DNA /RNA strands
Buffer solution - provide optimum pH for reactions to occur in

20
Q

3 stages of PCR?

A

DENATURATION: the double-stranded DNA is heated to 95°C - breaks the H bonds between 2 strands

ANNEALING – temp decreased to 50 - 60°C so that primers (forward and reverse ones) can anneal to the ends of the single strands of DNA

ELONGATION/EXTENSION– temp increased to 72°C for at least a minute, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules

PCR occurs in thermal cycle - provides optimal temp at each stage/controls length of time at each stage

21
Q

Why is TAQ polymerase used in PCR?

A
  1. Comes from thermophilic bacterium - doesn’t denature at high temps
  2. It’s optimum temp is high enough to prevent annealing of DNA stands that have not been copied yet
22
Q

What is electrophoresis?

A

Process used to separate proteins or DNA fragments of different sizes
- molecules separated according to size/mass and their net charge

23
Q

Why does electrophoresis work?

A

Electrical charge : + molecules will move towards CATHODE (-) and entire molecules mov towards ANODE (+)
- DNA is - charged bc of the phosphate - molecules move towards anode

Different sizes: tiny pores in gel mean smaller molecules move quickly /larger molecules move slower through the gel

Type of gel: different gel have different sized pores which affect sped at which molecules can move through them

24
Q

How to prepare DNA for sequencing /analysing ?

A

First amplify (increase number) of DNA molecules by PCR
- use restriction endonucleases to cut DNA into fragments
- different restriction enzymes cut the DNA at different base sequences - use enzymes that will cut close to the VARIABLE NUMBER TANDEM REPEAT regions (VNTR) - found in non coding part of DNA
- contain variable numbers of repeated DNA sequences /vary between different ppl

25
Q

Method of electrophoresis ?

A
  1. Create an agarose gel plate in a tank. Wells are cut into the gel at one end
  2. Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the tank
    Insert the fragments into the wells using a micropipette
  3. Apply an electrical current to the tank. The negative electrode must be connected to the end with the wells as DNA fragments will move towards the anode
  4. The smaller mass / shorter pieces of DNA fragments will move faster and further from the wells than the larger fragments
26
Q

How to make fragments visible in electrophoresis ?

A
  • must be transferred onto absorbent paper or nitrocellulose + heated to separate the two DNA strands
  • Probes are then added, after which an X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which is compared to a control fragment of DNA
27
Q

What are probes in electrophoresis?

A

Single stranded DNA sequences that are complementary to the VNTR regions
- contains a means by which to be identified:
A RADIOACTIVE LABEL: probes emit radiation that makes X-RAY film go dark, creating a pattern of dark bands
A FLUORESCENT STAIN/DYE : fluoresces when exposed to uv light - create pattern of coloured bands

28
Q

How are proteins prepared for electrophoresis?

A
  • different amino acids (because of the different R groups) determine the charge
    The charge of the R groups depends on the pH/ therefore buffer solutions used during the separation of proteins to keep the pH constant

Proteins prepared for electrophoresis by:
Denaturing (to break the disulfide bonds)
- Then manipulating the proteins into rod shapes (which are negatively charged) to allow separation by size

29
Q

How can gel electrophoresis be used to show genotypes?

A

By separating polypeptide chains produced by different alleles

30
Q

How do scientists create a DNA profile?

A
  1. Obtain DNA - from root of hair, blood,semen, saliva
  2. Use PCR to produce large quantities of required fragment of DNA
  3. Use restriction endonucleases to cut amplified DNA molecules into fragments
  4. Separate fragments using gel electrophoresis
  5. Add radioactive/fluorescent probes that are complementary /bind to specific VNTR regions
  6. X-ray images produced /UV lights used to produce images of fluorescent labels - pattern of bars (DNA profile ) to be analysed
31
Q

Use of DNA profiling?

A

Forensic med/criminal investigations :
Identify suspects for crimes

  1. Samples of body cells or fluids taken from the crime scene or victims body (eg. rape victims)
  2. DNA is removed and profiled
  3. The profile is compared to samples from the suspect , victim and people with no connection to the crime (control samples)
  4. Care must be taken to avoid contamination of the samples
  • also used to identify bodies /body parts that are unidentifiable
  • risk of developing particular diseases - certain VNTR associated with increased risk of cancers/heart disease
  • determine familial relationships - who the father is/immigration cases (see if family are related)
  • species conservation - reduce chances of inbreeding

Biology Alevel (24 decks)

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