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First year B > L35-40: DNA Technology > Flashcards

L35-40: DNA Technology Flashcards

1
Q

What is PCR?

A

PCR is used to amplify a single sequence of DNA into many more identical copies in vitro.

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2
Q

Requirments for PCR

A
  • DNA Template
  • DNA Primers
  • Thermostable DNA polymerase
  • dNTPs- deoxyribonucleoside triphosphates
  • Magnesium
  • Thermocycler
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3
Q

Role in PCR: DNA Template

A

Small source of DNA providing code to be copied.

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4
Q

Role in PCR: DNA Primers

A

primers/Oligonucleotides are single stranded containing 15-20 nucleotides.

-They anneal to opposite ends of section being copied

Therefore we have a ‘forward primer’ and a ‘reverse complement primer’

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5
Q

Role in PCR: Thermostable DNA polymerase

A

is stable at 95C and adds dNTPs to 3’ end of primers in the new strand to grow the chain.

*Requires magnesium

Examples:
– Taq polymerase
– Pfu polymerase
– Vent polymerase

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6
Q

Role in PCR:

dNTPs- deoxyribonucleoside triphosphates

A

Used to produce new strand of DNA

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7
Q

Role in PCR: Magnesium

A

Required by the thermostable DNA polymerase

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8
Q

Role in PCR: Thermocycler

A
  • Denaturation (94C)
  • Primer annealing (60C)
  • Polymerisation ( (72C)
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9
Q

Describe the stages involved in PCR

A

Denaturation:
DNA heated to 94C in order to separate original DNA strands.

Primer annealing:
Sampled cooled to 60C and complementary primers are added. The cooler temperature allows Bond to for between primers and anneal to each strand.

Polymerisation:
Sample heated to 72C. Thermostable DNA polymerase is added and attaches complementary free DNA nucleotides to the 3’ ends of the new strands.

Number of original molecules has been doubled. The process can be repeated to continue to amplify DNA and make more copies.

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10
Q

What is ‘Reverse transcriptase-PCR’

A

Making DNA from an RNA template

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11
Q

What is Gel Electrophoresis used for

A

Separate biological molecules based on physical characteristics

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12
Q

What characteristic causes DNA molecules to be separated using Gel Electrophoresis

A

Separate DNA by size

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13
Q

Basic concepts of Gel Electrophoresis

A
  • DNA has a negative charge, thus can move with electric current (electrophoresis)
  • Use electric current to pull through porous substance (argos gel)
  • Shorter DNA goes faster, and therefore goes farther in a given time
  • Ladder with a known size used to compare DNA
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14
Q

How can DNA be visualised during Gel electrophoresis

A

Ethidium bromide (a mutagenic agent) is intercalated into DNA, this is fluoresces under UV light

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15
Q

DNA Sequencing

A

Exploits the principle of complementary base paring to allow the complete nucleotide sequence of a DNA molecule to be determined

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16
Q

Sanger method

A

Chain termination sequencing:

PCR but the polymerisation step uses terminator bases, these bases attach to the sequence being copied and at this point PCR is stopped, each type of bases is tagged with a fluorescent colour, this allows the point at which sequencing stops to be identified. The sample of DNA is therefore sequenced in fragments.

The fragmented PCR sample is run on an electrophoresis gel, allowing the bases attached at different lengths to be identified.

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17
Q

Next generation sequencing

A

Type=Sequencing by synthesis

DNA fragments are amplified using PCR adding one base at a time, however all bases used have a florescent colour that is removable. An electronic monitor captures an image of the amplified sequences and identify the bases added in real time. the fluorescent dye is removed and the cycle Is repeated multiple times, the electronic motitor identifies the colour change at each ‘dot; and thus the base sequence can be determined.

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18
Q

Third generation sequencing

A

Single strand of DNA moved through a very small pore in a membrane and the bases are identified individually due to fact each base interrupts an electrical current by a different amount of time and can thud be identified.

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19
Q

Describe recombinant DNA technology

“DNA cloning”

A

A gene from an organism is inserted into a microorganism using a vector, this causes the microorganism to produce the proteins which the gene coded for.

  • produce more of target product
  • secrete product into surrounding medium
  • not survive in external environment
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20
Q

Requirements for recombinant DNA technology

A
  • Donor cells: with required gene
  • Restriction endonuclease: to cuts gene from donor cell and cuts plasmid open
  • Vector: plasmids or artificial chromosomes to carry DNA from donor to host.
  • DNA ligase: to seal DNA fragments into plasmid
  • Host cells: to receive the altered vector and produce more DNA
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21
Q

How does restriction endonuclease work

A

They are enzymes which recognise only one specific sequences of DNA and cut them into fragments at the restriction site producing DNA fragments with “sticky ends” (unpaired nucleotides at each end).

*used to cut specific genes out of a chromosome and to cut open plasmids.

If same enzyme is used to cut out the required gene and cut open the plasmid they will have complementary sticky ends; this allows the gene to be inserted into the plasmid.

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22
Q

Artificial chromosomes

A

Made by adding non-baterial DNA to bacterial chromosomes.

*can carry larger DNA fragments than plasmids

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23
Q

Required qualities of vectors

A
  • Restriction sites
  • Regulatory sequence
  • Selectable markers
  • Origin of replication
  • safety mechanism
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24
Q

Vector: restriction sites

A

Contain target sequence of bases that can be cut open by th same restriction endonuclease used to cut the donor DNA.

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25
Q

Vector: regulatory sequence

A

Controls gene expression

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26
Q

Vector: selectable markers

A

Marker genes to indicate whether host cell has taken up vector.

  • Antibiotic resistance, cell grown in medium with antibiotic, will therefore only survive if host has taken up the vector.
  • Gene coding for fluorescent proteins which can be identified using a microscope.
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27
Q

Vector: orgin of replication

A

Allow plasmid or chromosome to self-replicate.

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28
Q

Vector: Safety mechanisms

A

Genes introduced to preven the survival of microorganisms in an external environment.

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29
Q

Steps required in recombinant DNA technology

A
  • Identification: of gene of intrest in donor
  • Isolation: of gene of interest
  • Insertion: of gene into vector
  • Transformation: vector inserted into host cells
  • Expression: of introduced gene in host.
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30
Q

Recombinant DNA Technology: Limitations of Eukaryote DNA

A
  • include both introns + exons, therefore primary transcript is modified by spicing and undergoes post translational modification.
  • Plant and animal cells produce proteins which are inactive in bateria . However recombiant yeast cells are successful.
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31
Q

Gene Expression

A

The process by which information encoded in DNA directs the synthesis of proteins or, in some cases, RNAs that are not translated into proteins and instead function as RNAs

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32
Q

Gene Expression analysis

A

This is the identification of a gene which is produced by cells of interest.

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33
Q

Nucleic acid hybridisation

A

-Two single stranded nucleic molecules with complementary bases form a double stranded molecule.

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34
Q

Gene expression analysis: Give a description of an example of
In-situ Hybridisation

A

Used to determine if a gene is expressed by a cell of interest.

Say we have a cloned a gene which is suspected to have an important role in embryonic development, and we wish to identify which embryonic cell expresses this gene.

First we must identify the mRNA which is produce by the gene. The mRNA can be detected by nucleic acid hybridisation with a ‘nucleic acid probe’ this is a short, single-stranded nucleic acid molecule which is complementary to the target mRNA. Nucleic acid probes can be synthesised from the cloned gene, thus they will be complementary to the mRNA, during synthesis they are tagged with a fluorescent dye so can be identified.

A solution containing these probes is applies to the embryos, the probe molecules hybridise complementary sequences on the mRNA within the embryonic cells in which the gene id being transcribed.

The embryo can then be viewed using fluorescence microscopy to identify where the gene is expressed in the mRNA.

35
Q

Gene expression analysis: Give a description of an example of Reverse-Transcriptase PCR
(RT-PCR)

A

-Denaturation: mRNA incubated with poly-dT primer

hight temp -> single stranded
cooler temp -> poly-dT anneals

  • Addition of reverse-transcriptase + dNTPs + Buffer
  • > hybridisation occurs
  • The mRNA is degraded
  • Complementary single stranded DNA remains
  • second strand of DNA is synthesised using DNA polymerase and complementary bases
  • Complete cDNA with no introns is synthesised

This process is carried out for mRNA sample collected from different genes this allows for comparison of the amount of mRNA within different genes to be determined.

To analyse timing of expression of gene, take mRNA samples from different stages of development and use reverse-transcriptase PCR to synthesis cDNA, (note primer used is complementary to gene of interest, therefore only mRNA that contains gene of interest will produce amplified)

Gel electrophoresis of cDNA, amplified regions observed as bands, indicates cDNA synthesised from mRNA contained gene of interest.

36
Q

When would it be preferred to use RT-PCR not southern blotting

A

When you wish to make a comparison off the amount of a specific mRNA present in different samples at the same time.

37
Q

Gene expression analysis: Give a description of an example of Quantitate Real-time PCR
(q-PCR)

A

Measure rate of PCR product production to estimate starting amount.

Fluorescent dye that fluoresces only when bound to double stranded PCR product.

38
Q

DNA Microarray Analysis

A

A method used to detect and measure the expression of thousands of genes at the same time.

Reverse-Transcriptase PCR using fluorescent bases to produce labelled cDNAs

Tiny amounts of a large number of single stranded DNA fragments representing different genes are fixed to a glass and tested for hybridisation with samples of labelled cDNA.

39
Q

RNA Sequencing

A

A method of analysing large sets of mRNA that involves making cDNAs and sequencing them.

-mRNA are isolated from tissue being studied and cut into small fragments. The mRNA fragments are reverse-transcribed into cDNAs, these cDNA are then sequenced. The sequences are then mapped by a computer onto the genome, the data indicates which sequences are expressed in a given tissue and the number of times the sequence is present.

40
Q

Gene expression modification

A

Altering the genetic material of cells in a predictable way.

41
Q

How genes can be studied analysing gene expression modification:
In-vitro mutagenesis

A
  • A gene is isolated, cloned and introduced to specific mutations.
  • Mutated gene is reintroduced to the cell and disables the non-mutated copies of that gene within the cell.
  • If the mutated gene destroys the function of the gene product a change in phenotype could help reveal the function of the disabled normal gene.
42
Q

How genes can be studied analysing gene expression modification:

CRISPR-Cas9

A

-Clustered Regularly Interspaced Short
Palindromic Repeats

  • Naturally occurring as a a bacteria protein which protects bacteria against bacteriophages, CAS9 bind to an RNA molecule, this RNA molecule will direct the CAS9 to cut a DNA sequence (CAS9 is capable of cutting any DNA sequence to which it is directed).

This function can be exploited by injecting a CAS9 which is already bound to RNA complex into a cell, the chosen RNA complex is engineered to target a specific gene, the complex binds to this gene, causing the CAS9 to cut both DNA strands of the target gene.

The cell can repair the broken DNA strand:

Method 1) Repair enzymes rejoin the ends of the broken DNA, sometimes inserting or deleting random nucleotides, this makes the gene unfunctional and allows researchers to study what the gene does by determining what function is missing as a result of knocking out the gene.

43
Q

Give another use of CRISPR-Cas9

A

CAS9 is capable of cutting any DNA sequence to which it is directed.

This function can be exploited by injecting a CAS9 which is already bound to RNA complex into a cell, the chosen RNA complex is engineered to target a specific gene, the complex binds to this gene, causing the CAS9 to cut both DNA strands of the target gene.

The cell can repair the broken DNA strand:
method2) When there is a remaining undamaged normal copy of the gene, CRISPR-Cas9 repair enzymes use this normal gene as a template and synthesise the correct gene sequence, this is used for gene therapy.

44
Q

Cloning

A

Product is genetically identical to the parent

45
Q

What is Organismal Cloning

A

Produces one or more organisms that are genetically identical to the parent

46
Q

Cellular differentiation

A

A process by which unspecialised cells become alterend to preform a special function.

47
Q

Meristems

A

A region of unspecialised cells in a plant that can divide to produce many copies of themselves and/or differentiate into specialised plant cells (such as xylem or phloem).

48
Q

Stem cells

A

Unspecialised cells in animals that can divide to produce many copies of themselves and/or differentiate into specialised animal cells (embryonic or adult tissue stem cells)

49
Q

Adult tissue stem cell

A

Not all genes in adult tissue stem cells can be switched on, consequently adult tissue stem cells can only differentiate into types of cells found in a particular tissue type (blood stem cells in marrow bone can give rise to all types of blood cell). These cells are described as multipotent.

50
Q

Embryonic stem cell

A

All genes in an embryonic stem cell can be swithched on, consequently embryonic cells can differentiate into any type of cell that make up the organism. These cells are described as pluripotent.

51
Q

multipontent

A

can produce limited number of cell types- for example cells belonging to a particular tissue type and act as a repair system to replace old/damaged cells

52
Q

pluripotent

A

All genes can be switched on so they can differentiate into most cell type within a body (can give rise to Embryo)

53
Q

totipotent

A

All genes can be switched on so they can differentiate into any cell type within a body
(can give rise to both the placenta and the embryo)

54
Q

Interest of cloning in relation to stem cells

A

If all the cells of an organism have the same genes then totipotent and cloning is possible but if cells lose genes during cell differentiation then cloning isn’t possible.

55
Q

Plant cloning, method to determine if possible

A

Differentiated cells found in root of carrot, incubated in culture medium could grow genetically identical adult plants.

Provides evidence that plant cells can ‘dedifferentiate’ into totipotent stem cells and produce clones.

56
Q

Animal cloning

A

nuclear transplantation-> remove nucleus of an egg and replace with nucleus of a less differentiated cell.

If the nucleus from the less differentiated donor cell retains full genetic compatibility then should be able to direct development of the recipient cells into all tissues and organs of the organism.

However very small percentage of nuclear transplantations develop to birth, since the potential of the nucleus decreases with age = more difficult!

57
Q

Application of DNA Technology: Stem Cells

A

-Therapeutic uses of stem cells

Used to repair corneal damage: stem cells from the edge of a persons own cornea can be used to grow new cornea tissue

Treat burn victims: Persons own skin stem cells can be taken to grow temporary skin to make skin grafts on burns.

-Use during Drug testing
Stem cells can be used in drug testing to reduce the need for animal testing

58
Q

Application of DNA Technology: PCR

A
  • Allows for small mummified samples or fossil samples to be used for phylogenetic; this is the analysis of evolution to map when species diverged from one another by how much their DNA differs due to mutations.
  • Tissue typing for organ transplantaion
  • Forensics: tiny sample of DNA found at crime scene can be amplified to provide enough material for genetic fingerprinting.
59
Q

Application of DNA Technology

A

DNA cloning and synthetic biology to produce drugs (e.g., human insulin from bacteria)

60
Q

Induced pluripotent stem cells

A

Return differentiated cell to pluripotent state, however still has some differences from embryonic stem cells.

61
Q

Transdifferentiation

A

Turn differentiated cell directly into other differentiated cell type

62
Q

Purpose of Forensic Analysis of DNA

A
  • Identify suspects from evidence at crime scene
  • Identify victims of catastrophe
  • Identify familial relationships
  • Detect presence/species of microorganisms
  • Identify materials from endangered species (poaching
63
Q

Name Three Forensic Techniques

A
  • Restriction Fragment Length Polymerisation
  • Short Tandem Repeat
  • Sequencing
64
Q

Genome

A

Genome

An organism’s entire hereditary information encoded in DNA.

65
Q

Genes

A

A sequence of DNA that codes for proteins

66
Q

Genomics

A

A study of genomes

67
Q

Human Genome project

A

To determine the complete nucleotide sequence of each human chromosome.

68
Q

Genomic sequencing

A

The determination of the sequence of nucleotide bases for individual genes or entire genomes.

69
Q

Shotgun sequencing

A
  • DNA randomly cut into fragments
  • Fragments cloned in vectors (NOW THIS STAGE IS UNNECESSARY)
  • Each fragment sequenced
  • Bioinformatics is used to put the DNA fragments ‘back togther’ this is done by looking for overlaps in the sequence.
  • The fragment sequences are arranged into one overall sequence using the computer.
70
Q

Nucleotide Sequence Databases

A

All published DNA sequences must be submitted to one of these databases:

  • GenBank (USA),
  • EMBL (European)
  • Nucleotide Archive (UK),
  • DDBJ (Japan)

All three databases share each others data

71
Q

Gene annotation

A

Analysis of genomic sequences to identify protein coding genes and determine the function of their proteins.

Three stage process:

1) Computer annotation
2) Human curation
3) Experiments

72
Q

Gene annotation: computer annotation

A

-Search genomic sequence for features related to genes
(promoter regions, RNA polymerase binding sites, translation start/stop sites, etc.)

-identify hypothetical gene by comparison of it’s genomic sequence with known RNA sequences

73
Q

Gene annotation: Human Curation

A

Annotator views computer predictions and evidence and makes an assessment.

74
Q

Gene annotation: Experiments

A

Required to confirm gene expression and function

  • RNA-seq to confirm that it is expressed!
  • knockout/modification to probe function
75
Q

What is BLAST?

A

Basic Local Alignment Search Tool is a program that compares sequences

76
Q

Assumption which BLAST is based on

A

– If sequences are similar, they are likely to be related by common descent (homologous).
– Homologous sequences are likely to have similar functions.

77
Q

Systems biology

A

Aims to model the dynamic behaviour of whole biological systems based on a study of the interactions among the systems parts.

78
Q

What is GWAS

A

Genome-Wide Association Studies

79
Q

Genome size

A

Eukaryotes have larger genomes than prokaryotes

*There is no other clear relation between organism and genome size

80
Q

Compare number of genes within different types of Eukaryotes.

A

Multi-cellular eukaryotes have more genes than unicellular eukaryote

81
Q

What are Pseudogenes

A

gene sequences that have accumulated

mutations and are non-functional

82
Q

What is Repetitive DNA

A

DNA sequences found in multiple copies in the genome

83
Q

Transposable Elements

A

A segment of repetitive DNA from a section of chromosome that can move within the genome by means of DNA and RNA intermediate

84
Q

multi-gene families

A

A collection of genes with similar or identical sequences, presumably of common origin

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