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A-level Biology > 6.3 Manipulating genomes > Flashcards

6.3 Manipulating genomes Flashcards

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1
Q

What is a genome?

A

all the genetic information within an organism

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2
Q

What does BAC stand for

A

Bacterial Artificial Chromosomes

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3
Q

How are genomes sequenced?

A

Samples of genomes are sheared into smaller sections
These are transferred to BACs
and transferred to coli cells.
As these cells grow in culture, many clones of the sections are produced - clone libraries

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4
Q

What is PCR?

A

Polymerase Chain Reaction

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5
Q

What is Sanger Sequencing?

A

Also called chain termination
Uses Modified nucleotides (dideoxynucleotides), which do not allow another nucleotide to join them after the sequence - terminating the process.
This results in scientists knowing the terminal base in a sequence.
As each test tube has one type of base in, scientists can then put the base sequence through gel electrophoresis and find the base sequence.

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6
Q

What else is there in Sanger sequencing?

A

A radioactive primer on each deoxynucleotide

RNA polymerase solution kept at optimum temperature

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7
Q

What is the aim of PCR?

A

To produce large quantities of DNA from very small quantities

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8
Q

What are primers?

A

Short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied. They define the region that is to be amplified by identifying to the DNA polymerase where to begin building the new strands

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9
Q

How does PCR work?

A
  1. Double-stranded DNA is heated to 95 so the H-bonds between bases denature and the strands separate to 2 singles
  2. Annealing - the temperature is decreased to 50 so primers can anneal to the exposed complimentary bases at the 3’ end of DNA
  3. Elongation - temperature increased to 72 to allow taq / DNA polymerase to attach to nucleotides along each DNA strand - forms phosphodiester bonds between sugar and phosphate group
    Results in two new DNA fragments which are copies of each other
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10
Q

What are advantages of PCR?

A

Very rapid - billions of copies can be made in hours
Does not require living cells - only required one base sequence - no culturing

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11
Q

What is the purpose of Electrophoresis?

A

To separate DNA sequences by weight/length and overall charge

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12
Q

How is DNA separated and prepared for electrophoresis?

A
  1. Increase the number of DNA molecules by PCR
  2. Restriction endonucleases to cut DNA into fragments
    –> Different RE’s will cut the DNA at different base sequences
  3. Use RE’s which cut close to the variable number tandem repeat regions
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13
Q

What are VNTRs?

A

Variable Number Tandem Repeat regions
—> Found in non-coding part of DNA
—> They contain variable numbers of repeated DNA sequences and are known to vary between different people (except for identical twins). These VNTR may be referred to as ‘satellite’ or ‘microsatellite’ DNA

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14
Q

What is the method for Gel Electrophoresis?

A
  1. Submerge the gel in electrolyte solution in the tank
  2. DNA fragments are placed in a well at the top of the gel
  3. Apply electrical current - the DNA fragments will then move towards the anode (positive pole) due to the attraction between the negatively charged phosphates of DNA and the anode
  4. The smaller mass / shorter pieces of DNA fragments will move faster and further from the wells than the larger fragments
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15
Q

How are the DNA fragments observed after Electrophoresis?

A

The fragments are not visible so must be transferred onto absorbent paper or nitrocellulose which is then 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 generally compared to a control fragment of DNA

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16
Q

What are probes?

A

Single-stranded DNA sequences that are complementary to the VNTR regions sought by the scientists - they can be identified:
1. Radioactive label
2. Fluorescent dye

17
Q

Why is DNA profiling used?

A

Enables scientists to identify suspects for a crime and identify corpses
—> every person (apart from identical twins) has repeating short non-coding regions of DNA (20 to 50 bases) that are unique to them, they are called variable number tandem repeats (VNTRs)

18
Q

How is a DNA profile created?

A
  1. Obtain DNA - hair, blood, saliva
  2. Use PCR to increase the quantity of DNA
  3. Restriction endonucleases to cut the DNA into fragments
  4. Separate fragments using electrophoresis
  5. Add radioactive/fluorescent probe which is complementary and binds to specific VNTR regions
  6. Analyse the Bar pattern (DNA profile)
19
Q

Other uses of DNA profiling?

A
  1. identify individuals that are at risk of developing particular diseases, as research shows that certain VNTR sequences are associated with an increased incidence of particular diseases eg. cancers and heart disease
  2. determine familial relationships for paternity cases (to suggest who the father is) or immigration cases (to determine if the family are related)
  3. species conservation to help scientists with captive breeding programmes to reduce chances of inbreeding
20
Q

What does Genetic engineering mean?

A

the manipulation of the DNA sequences of an organism

21
Q

What features of the genetic code allow for genetic engineering

A

It is universal - all have the same 4 nitrogenous bases
the same codons code for the same amino acids in all living things

22
Q

What are the key steps of genetic engineering

A
  1. Isolation of DNA containing required gene
  2. Insertion of DNA into vector
  3. Transformation: Transfer of DNA into a suitable host
  4. Identification - identify the host containing the vector and DNA using markers
  5. Cloning of most successful hosts
23
Q

How is the DNA containing the required gene isolated?

A

Method 1: Restriction endonucleases cut sticky ends on a palindromic recognition site of the DNA
Method 2: mRNA and reverse transcriptase and ligase cuts blunt ends
(no introns, all coding DNA)

24
Q

How is DNA inserted into a vector?

A

Plasmid from a bacteria is cut with the same restriction endonucleases as the gene
The sticky ends are joined together using DNA ligase
Forms recombinant DNA within the bacterium

25
Q

How is the plasmid which contains the DNA identified?

A

Use of gene markers
1. Fluorescent markers are incorporated into the plasmid
2. If the DNA fragment has been successfully inserted, then the bacteria which do not fluoresce contain the DNA (as the DNA is inserted into the fluorescent gene on the plasmid)
3. It can be identified under UV light

26
Q

How is the bacteria cloned?

A

In Vivo: within a living organism

27
Q

What are advantages to using genetically modified micro-organisms to produce recombinant human proteins?

A

Cost-effective
Faster
Simpler
Reliable supply available
Proteins made will be identical to human proteins
Overcomes ethical or religious reasons to not use pork / animal induced proteins

28
Q

Ethical issues with GM plants:

A

Advantages
Golden Rice - no vitamin A deficiencies
Maize - can survive in harsh conditions so a greater yield
Therapeutic antibodies from GM tobacco plants can treat rabies and save lives

Disadvantages:
- LEDC’s become reliant on richer countries
- Are GM crops safe to eat
Resistance could be spread between species
- Farmers must buys seeds from a manufacturer

29
Q

What is Somatic cell therapy?

A

Replacing a faulty gene with a healthy allele in affected somatic cells

30
Q

What is Germ line cell therapy?

A

Inserting a healthy allele in germ cells or very early embryo

31
Q

How does Germ line cell therapy work?

A

The corrected gene is inserted into a fertilised egg via IVF
All cells of the embryo will contain the modified gene as the original cell divides by mitosis
Permanent and ensures offspring inherit the corrected gene
Illegal

32
Q

How does Somatic cell therapy work?

A

Copies of the corrected gene are inserted directly into the somatic cell of the sufferer
Does not prevent disease from occuring in the next generation, as it does not affect sperm/egg cells
Has to be repeated many times as results do not last long

33
Q

How are liposomes used as vectors?

A
  1. Functioning genes are isolated from human cells and inserted into plasmids
  2. Plasmids are extracted and wrapped in lipid molecules
    —> forms the liposome
  3. Liposome sprayed into patients airway via nasal spray
  4. Liposomes pass easily through plasma membrane of cells and nucleus
34
Q

How are viruses used as vectors?

A
  1. Grow harmless adenoviruses in epithelial cells
  2. Add recombinant plasmids which contain the functional gene
  3. Viruses are isolated from the epithelial cells and purified
  4. Virus sprayed into the nostrils via aerosol
  5. Virus injects DNA into epithelial cells of lungs

A-level Biology (16 decks)

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