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MTC > DNA technologies > Flashcards

DNA technologies Flashcards

wk 12

1
Q

What is PCR?

A

Technique that Amplifies single or few copes of DNA across several orders of magnitude

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2
Q

What are the three reactions for PCR?

A

DENATURATION: heat to 95 degrees to denature into two separate strands

ANNEALING: cooling to allow primers to bind to template

EXTENSION: Temperature increased to optimum temp for DNA polymerase

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3
Q

What is SYBR Green?

A

= double stranded DNA-binding dye that intercalates into double-stranded DNA (dsDNA)

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3
Q

What are the two methods of choice for nucleic acid quantification?

A

PCR and qPCR

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4
Q

What is SYBR Green used for?

A

A dye that allows for the measurement of the amount of PCR product.

As amplification proceeds = number of SYBR green molecules increase (fluorescence is proportional to amount of products accumulated.

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5
Q

What is a restriction enzyme?

A

Enzyme that cuts DNA at restriction site

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6
Q

What does PCR require for aplification?

A

Two primers (single strand DNA with complementary sequences to ends of DNA want to amplify)

Free dNTPs: deoxynucleotides triphosphates (building blocks of new strands)

Buffer: keep enzyme working

Magnesium ions: cofactor for enzyme

DNA polymerase: catalyses synthesis of new DNA strands

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7
Q

What is the old method used to measure PCRs?

A

PAGE - agarose gel

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8
Q

Describe the use of agarose gel in PCR meaurement.

A

It is used to visualise the PCR fragments

they pass electrical current through agarose gel

DNA (negative) moves towards positive terminal

Distance it moves is dependent on number of base pairs

More base pairs = heavier and slower movement

Compare movement against a ladder of DNA fragment lengths

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9
Q

Is DNA positive or negative?

A

negative

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10
Q

What is the new method for DNA amplification?

A

qPCR

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11
Q

what is a Palindromic sequence of DNA?

A

= sequence of one strand of DNA is the same as the other read backwards.

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12
Q

How do restriction enzymes and palindromic sequences create new strands of DNA?

A

Restriction enzyme cleaves DNA into two strands with overhanging sticky ends

e.g. EcoRI 5’GAATTC’3 and 3’-CTTAAG-5” : Pstl cuts between A and G and cretaes two four base 3’ overhangs

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13
Q

How is the plasmid in cloning vectors cut?

A

with a restriction enzyme

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14
Q

What makes up a Cloning Vector?

A

= small piece of DNA + plasmid (or cell of higher organism) + foreign DNA

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15
Q

Describe the DNA used in a cloning vector

A

The DNA must be from another organism (virus) and be stably maintained in an organism.

16
Q

describe the basic process of a cloning vector?

A

Plasmid cut with restriction enzyme

DNA template replicated with PCR (with primers of a specific restriction site)

Product is ‘glued’/ligated into an expression vector with matching site

The plasmid makes a protein –> overexpression

17
Q

What are three examples of the use of cloning vectors?

A

Pig insulin (old technique)

Recombinant human insulin production (new technique)

Humanised antibodies

18
Q

Outline the process of recombinant human insulin production.

A

Human gene isolated

Bacterial plasmid isolated and cut with restriction enzyme

Human insulin gene inserted using DNA ligase

Vector used to create recombinant bacterium

Bacterium grows in fermentation tank

Insulin extracted and purified

19
Q

What are the two humanised antibodies used as recombinant protein and therapy

A

Herceptin and Avastin

20
Q

What is Macular degeneration?

A

Degeneration of the retina (subretinal haemorrhage)

Can be seen in diabetes mellitus (high BGL = vascular endothelial growth factors cause blood vessel growth = lifted up retina, leak of blood and fluid and central vision loss.

21
Q

Discuss the role of Avastin on Macular Degeneration.

A

Slows progression of disease considerably

Avastin prevents VEGF from binding to the membrane and causing angiogenesis (growth of blood vessels)

22
Q

What is EGFR and what does its over-expression result in?

A

Transmembrane tyrosine kinase glycoprotein activated by EGF

Overexpression = tumours of breast, lung, colon, cervix, ovary, oesophagus and endometrium.

23
Q

what is the relationship between herceptin and cancer?

A

Provides a target for the immune system to destroy cancer cells that it is attached to

Lower metastases and cancer growth when used

24
Q

What us Herceptin?

A

Immune targeted therapy

25
Q

What does Herceptin and Avastin treatment prevent and what is the impact of this?

A

Herceptin and Avastin co-treatment prevents micro vessel and epithelium growth.

There is a worse prognosis for those with higher HER2/VEGF density

25
Q

What is an example of a virus mediated anti-cancer treatment and how (generally) does it work

A

T-VEC (engineered virus) provokes immune response to treat advanced melanoma

T-VEC = from herpes simplex virus-1, virus becomes attenuated so no longer causes herpes

26
Q

What is the old method of DNA sequencing?

A

Sanger method

27
Q

What does the Sanger method use?

A

A single stranded DNA template

DNA primer

DNA polymerase

Normal deoxynucleotides (dNTPs)

Modified nucelotides (dideoxyNTPs: ddNTPs) that terminate DNA strand elongation.

28
Q

Outline the Sanger Method

A

Four PCR mixes set up, containing stocks of normal nucleotides plus one dideoxynucleotide (ddA, ddT, ddC or ddG)

As a typical PCR will generate over 1 billion DNA molecules, each PCR mix should generate all the possible terminating fragments for that particular base

When the fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length

If a distinct radioactive or fluorescently labelled primer is included in each mix, the fragments can be detected by automated sequencing machines

29
Q

What are the three types of Next Generation Sequencing?

A
  • Illumina (Solexa) sequencing
  • Ion torrent: Proton / PGM sequencing
  • SOLiD sequencing
30
Q

What are the benefits of NGS?

A

Much more quicker cheaper

revolutionised study of genomics and molecular biology

allowed Genome Wide Association Studies
-detect rare disease
-personalise medicine
-better techniques for cancer treatment

31
Q

What are Genome Wide Association Studies and what do they allow for?

A

Rapid scanning makers across set of DNA = find genetic variations associated with a particular disease

Used to develop strategies to detect, treat and prevent disease

32
Q

What are SNPs?

A

Positions in genome where some individuals have one nucleotide, and others a different one

Swapped nucleotide may be a marker or elevate risk of disease

SNPs are found in: coding and non-coding regions

Very high frequency: 1 in 1000 to 1 in 300 SNPs

can act as a marker for a gene

MTC (14 decks)

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