Topic 4 - chapter 10 Flashcards Preview

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Flashcards in Topic 4 - chapter 10 Deck (38)
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1

What does gel electrophoresis do?

separates DNA fragments of different sizes when voltage is applied

2

Is DNA +vely or -vely charged?

-vely charged

3

What is hybridisation?

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

4

How does hybridisation basically work?

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

5

Which two techniques does Southern blotting combine?

electrophoresis & complementary basepairing

6

What are DNA probes used for?

to recognise a desired nucleotide sequence

7

Can recombinant DNA be copied inside bacterial cells?

yes

8

What can be used to clone DNA?

Specialised plasmid vectors

9

cDNA libraries represent what?

mRNA produced by a particular tissue or organ

10

What is PCR?

Polymerase chain reaction - used to amplify selected DNA sequences

11

Plasmids...

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

12

A genomic library contains...

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

13

What are positive clones and how can they be found?

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

14

What is a cDNA library? What does it contain?

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

15

From tissue (e.g brain) to cDNA copy...

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

16

Describe the PCR aloud...

slides 30 - 32...

17

What are some applications of PCR?

- 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)

18

Studying gene function usually involves what?

constructing recombinant DNA molecules with plasmid vectors

19

Understand the process of sequencing...

slides 38 - 40

20

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

- 'shotgun'
- 'clone by clone'

21

What is an 'expression vector'? What are it's implications?

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

22

What is 'shotgun' sequencing? What are its drawbacks?

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

23

What is clone-by-clone sequencing?

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

24

What is the 'eternal' pathway?

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

25

What are 2 common reporter proteins?

enzymes beta-galactosidase & green fluorescent protein (GFP)

26

What are reporter genes used for?

to monitor when & where a gene is expressed

27

What is & in situ hybridisation used for? Probes?

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

28

Some applications of in situ hybridisation method...

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

29

What is FISH?

Fluorescence in situ hybridisation

30

What are DNA microarrays used for?

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