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Genomics > Enzymes and Restriction Mapping > Flashcards

Enzymes and Restriction Mapping Flashcards

1
Q

Types of genetic engineering

A

Recominant proteins

Transgenic organisms

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2
Q

Examples of recombinant proteins

A

Insulin
Interferon
G-CSF

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3
Q

What is interferon?

A

Produced by cells that have been infected by viruses, induces anti-viral response in cells, given to patients with Hep C infection.

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4
Q

What is G-CSF?

A

Granulocyte-colony stimulating factor given to patients that are undergoing radiotherapy

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5
Q

Examples of use of transgenic organisms

A

Disease models

Improved agricultural yields

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6
Q

Nucleases

A

Degrade nucleic acids by hydrolysis (cleaving) phosphodiester bonds

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7
Q

Ribonuclease

A

RNAse - degrade RNA

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8
Q

Deoxyribonuclease

A

DNAse - degrade DNA

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9
Q

Exonuclease

A

degrade from end of molecule

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10
Q

Endonuclease

A

Cleave with nucleotide chain

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11
Q

Why is restriction important?

A

Limit transfer of nucleic acids from infecting phages (viruses) into bacteria.

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12
Q

What are restriction enzymes used for?

A

They were initially used to prevent the infection of bacteria by viruses (phages) infecting bacteria. Removing the viral DNA is done via a restriction enzyme that would cleave it into different pieces.

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13
Q

How was the use of restriction enzymes first identify?

A

First identified in E.coli and is called Restriction enzyme 1. EcoR1 binds and recognises a specific DNA sequence GAATTC.

It cleaves to form a 5’ overhang on both sides. This cleavage also leaves a phosphate group at the 5’ end of the molecule. On the 3’ end, a OH- group is left. This is specific to EcoR1.

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14
Q

What are restriction sites?

A

A 4-8 base pairs in length, depending on the enzyme, and palindromic.

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15
Q

Define palindromic

A

Can be read in both directions

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16
Q

How often does a 4 base recognition sequence occur?

A

4 to the power of 4 = 256 bases

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17
Q

How often does a 6 base recognition sequence occur?

A

4 to the power of 6 = 4096 bases

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18
Q

What do some nucleases produce when they cut sequences?

A

An overhang - EcoR1 produces 5’ and Kpn1 produces 3’ overhangs.

Some nucleases produce a blunt end - Alu1 recognises the DNA sequence and cleaves to produce blunt ends. It recognises 4 base pairs and digests the DNA.

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19
Q

What are restriction maps?

A

Map of restriction sites within a molecule where restriction enzyme sites are.

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20
Q

Why is restriction mapping used?

A

It is a crude way of mapping an unknown molecule when sharing data it can be used in other labs.
It is also useful way of describing plasmids and what they look like.

21
Q

How many restriction enzyme sites does linear DNA have?

A

Only have one restriction enzyme site.

22
Q

How many restriction enzyme sites does plasmid DNA have?

A

2 or more restriction enzyme sites.

23
Q

What is restriction enzymes done in molecular diagnostics?

A

Can be used in sickle cell anaemia - there is a mutation at codon 6 which causes a change in the amino acid.

24
Q

What can single nucleotide changes do? Give examples

A

It can create/destroy restriction enzyme sites. For example, in sickle cell anaemia, DdeI site (5’CTNAG3’) is lost to SCA. This means that a person with SCA has no DdeI site.

25
Q

Explain the process of genomic DNA testing

A

Genomic DNA can be purified from a fetus. This will result in a fragment and will be amplified by PCR. Cut using restriction enzymes and separated via gel electrophoresis.

26
Q

Fragments of SS (sickle cell anaemia) - related to SCA

A

Two fragments 376bp and 175bp

27
Q

Fragments of AS (carrier) - related to SCA

A

One copy of each gene therefore will have both 376bp, 67bp, 201bp and 175bp.

28
Q

Fragments of AA (normal) - related to SCA

A

201bp, 175bp and 67bp

29
Q

How are DNA molecules from different sources joined together?

A

By DNA ligase that can join human and/or bacterial DNA together.

30
Q

What does DNA polymerase need to work?

A

A primer

Deoxynucleotide 5’-triphosphates (dATP, dCTP, dGTP and dTTP). in order to extend the length of the DNA molecule

31
Q

What are the main uses of DNA polymerase?

A

PCR amplification
Generation of probes
Blunt-ending of DNA overhangs

32
Q

Why is DNA polymerases used?

A

So that two DNA fragments can be ligated.

33
Q

What does phosphatase do?

A
  • To hydrolyse a phosphate group off its substrate.
34
Q

What was calf intestinal alkaline phosphatase used for?

A

To better to work with in the lab in the past

35
Q

What was shrimp alkaline phosphatase used for?

A

Used more frequently now as can be produced in larger quantities

36
Q

Why is a phosphatase used when ligating strands?

A

To prevent cut plasmids from resealing (DNA ligase needs phosphate groups).

37
Q

What does a kinase do?

A

It produces phosphate from ATP and substrate

38
Q

What does polynucleotide kinase do?

A

It adds phosphate to 5’ hydroxyl group of DNA or RNA.

39
Q

Why is a polynucleotide kinase used?

A
  • To phosphorylate chemically synthesised DNA so that it can be ligated to another fragment.
  • To sensitively label DNA so that it can be traced.
40
Q

How is DNA traced?

A

By radioactively labelled ATP

Fluorescently labelled ATp

41
Q

What is a probe?

A

Fragment of ssDNA(or RNA)
20-1000 bases in length
Complementary to the gene of interest

42
Q

What expresses reverse transcriptase?

A

Only expressed in viruses with DNA genome

43
Q

What is reverse transcriptase?

A

It is a RNA dependent DNA polymerase
Present in retroviruses who make DNA from their RNA genome.
Makes DNA copies from RNA molecules
Isolated from RNA-containing retroviruses
Synthesises a DNA molecule complementary to a mRNA template using dNTPs

44
Q

What is the use of reverse transcriptase?

A

To check the expression levels of RNA/DNA in PCR

45
Q

Describe how reverse transcriptase works

A

RNA can be primed with random primers. Reverse transcriptase will bind to the cDNA and then convert it to mRNA.

46
Q

What are random primers?

A

cDNAs up to 700bp that will cover all of the length of all of the RNA molecules

47
Q

Why is oligo(dT) primers used?

A

They are useful for cloning cDNAs and cDNA libraries.
RNA is sometimes purified from cells, but may contain a polyadenylated tail, but the oligo (dT) primer will have an oligo T tail and allow reverse transcriptase to work.

48
Q

What are gene specific primers?

A

Primers designed to copy a specific gene of interest

Genomics (19 decks)

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