Topic 4 - chapter 10

Question 1

What does gel electrophoresis do?

Answer 1

separates DNA fragments of different sizes when voltage is applied

Question 2

Is DNA +vely or -vely charged?

Answer 2

-vely charged

Question 3

What is hybridisation?

Answer 3

a sensitive way to detect specific nucleotide sequences based on complementary basepairing

Question 4

How does hybridisation basically work?

Answer 4

DNA double helices + hight temp/high pH -> denaturation to single strands (H bonds break) + slowly cool/low pH -> renaturation restores DNA double helices

Question 5

Which two techniques does Southern blotting combine?

Answer 5

electrophoresis & complementary basepairing

Question 6

What are DNA probes used for?

Answer 6

to recognise a desired nucleotide sequence

Question 7

Can recombinant DNA be copied inside bacterial cells?

Answer 7

yes

Question 8

What can be used to clone DNA?

Answer 8

Specialised plasmid vectors

Question 9

cDNA libraries represent what?

Answer 9

mRNA produced by a particular tissue or organ

Question 10

What is PCR?

Answer 10

Polymerase chain reaction - used to amplify selected DNA sequences

Question 11

Plasmids…

Answer 11

are circular extrachromosomal pieces of DNA with an origin of replication and do NOT contain essential genes

Question 12

A genomic library contains…

Answer 12

all DNA from an organism packaged into small fragments cloned into plasmids

Question 13

What are positive clones and how can they be found?

Answer 13

positive clones are desired clones containing part or the whole gene of interest and can be found via hybridisation

Question 14

What is a cDNA library? What does it contain?

Answer 14

a representation of the mRNA produced by a certain tissue

contains only expressed sequences - so <1.5% of the human genome that are coding sequences

Question 15

From tissue (e.g brain) to cDNA copy…

Answer 15

1. Tissue cells (brain) 2. lyse & purify mRNA 3. mRNA with poly A tail at 3’ end 4. hybridise with poly T primer 5. make DNA copy with reverse transcriptase 6. degrade RNA with RNase H 7. synthesise complimentary DNA strand using DNA polymerase with 5’ end of original mRNA acting as primer 8. double-stranded cDNA copy of original mRNA

Question 16

Describe the PCR aloud…

Answer 16

slides 30 - 32…

Question 17

What are some applications of PCR?

Answer 17

• amplifying DNA strands
• 1st step in cloning process (genomic/DNA clones or cDNA/mRNA)
• very sensitive detection system/diagnostic method (HIV eg)
• very sensitive identification method with extremely high resolution (short tandem repeats (STR) locus in a single individual - maternal & paternal/paternity tests with electrophoresis)

Question 18

Studying gene function usually involves what?

Answer 18

constructing recombinant DNA molecules with plasmid vectors

Question 19

Understand the process of sequencing…

Answer 19

slides 38 - 40

Question 20

What are the 2 automated sequencing approaches used in human genomes?

Answer 20

• ‘shotgun’

- ‘clone by clone’

Question 21

What is an ‘expression vector’? What are it’s implications?

Answer 21

Specialised vectors used in DNA cloning that contain appropriate transcription & translation signals so that inserted gene is expressed at very high levels

Question 22

What is ‘shotgun’ sequencing? What are its drawbacks?

Answer 22

sequencing (determining the order of nucleotides) method where genome is broken into smaller, overlapping fragments then sequenced and genome assembled based on overlapping sequences
Drawbacks - only good for sequencing small genomes

Question 23

What is clone-by-clone sequencing?

Answer 23

technique where the human genome is broken into 100-200kb fragments -> into BAC -> E. coli -> divide (with BACs) -> many overlapping cloned fragments -> use restriction enzyme to generate ‘signature’ of each clone -> use shotgun method to determine nucleotide sequence of each BAC seperately -> assemble whole genome by stitching together the sequences of 1000s of BACs

Question 24

What is the ‘eternal’ pathway?

Answer 24

Recombinant DNA technique that begins with a protein of unknown function -> isolate gene encoding it -> churn out ++ protein for study of structure & activity

Question 25

What are 2 common reporter proteins?

Answer 25

enzymes beta-galactosidase & green fluorescent protein (GFP)

Question 26

What are reporter genes used for?

Answer 26

to monitor when & where a gene is expressed

Question 27

What is & in situ hybridisation used for? Probes?

Answer 27

to monitor the time & place mRNA product of a gene is expressed (good if gene's final product is RNA rather than protein) Probes include nucleic acid labeled with fluorescent dyes or radioactive isotopes

Question 28

Some applications of in situ hybridisation method...

Answer 28

- detect the presence of a virus in the cell eg. human papillomavirus (HPV) - embryonic development - locate genes on chromosomes (chromosome 5)

Question 29

What is FISH?

Answer 29

Fluorescence in situ hybridisation

Question 30

What are DNA microarrays used for?

Answer 30

to allow RNA products of thousands of genes to be monitored simultaneously -> identify & study complex gene expression patterns underlying cellular physiology -> which genes turned on/off as cells grow, divide or respond to hormones, toxins or infection

Question 31

How does DNA microarray work on a basic level?

Answer 31

mRNA extracted -> convert to cDNA & label with fluorescent dye -> mix & hybridise to microarray -> wash -> scan & combine images

Question 32

What is site-directed mutagenesis? Applications...

Answer 32

Taking a cloned gene and making a mutation in it in vitro (by changing single amino acid) to determine which parts of the polypeptide chain are crucial to fundamental processes like protein folding, enzymatic catalysis ie determine biological roles of each part of a given protein

Question 33

What are the 3 ways animals can be genetically altered?

Answer 33

Gene replacement - only mutant gene is active Gene knockout - no active gene present Gene addition - both genes are active

Question 34

What is the most modern method of genetic alteration?

Answer 34

Knockout mice using embryonic stem (ES) cells

Question 35

What is the knockout mouse factor that is responsible for premature aging?

Answer 35

helicase knockout - Xpd gene

Question 36

What is RNA interference (RNAi)?

Answer 36

utilises double stranded RNA molecule (whose nucleotide sequence matches that of the gene to be inactivated) inside E. coli that is typically used in worm (C. elegans) either fed or injected into it which is recognised as 'foreign' and is degraded into smaller fragments that can be passed onto progeny cells thus RNAi can cause heritable changes in gene expression

Question 37

How are transgenic plants important?

Answer 37

Important in cell biology (isolating receptors for growth regulators & analysing mechanisms of morphogenesis & gene expression in plants) Important in Ag (modify ratio of lipid, starch, protein in seeds to impart pest & virus resistance to plants or create modified plants tolerant to extreme habitats)

Question 38

Describe the process of generating transgenic plants using recombinant DNA techniques...

Answer 38

disc cut out of leaf -> incubated in Agrobacterium (carries recombinant plasmid with selectable marker & desired engineered gene) for 24hrs -> wounded cells release substance attracting bacteria which inject DNA into those cells -> only plant cells that uptake appropriate DNA & express selectable marker gene survive, proliferate & form callus -> growth factors induce shoot formation -> transfer to root-inducing medium -> grow into adults carrying engineered gene