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Locating and sequencing genes. DNA probes. Used to locate a specific gene sequence / gene A short, single stranded section of DNA… Labeled with… What do you think a probe could be labeled with?

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Presentation Transcript
dna probes
DNA probes
  • Used to locate a specific gene sequence / gene
  • A short, single stranded section of DNA…
  • Labeled with…
  • What do you think a probe could be labeled with?
  • Radioactive isotope – nucleotides are made using 32P phosphate. The radioactivity is detected using a photographic plate i.e. you can see where the probe is, and therefore can locate where the gene is…
  • Or the probe can be labeled with…
  • Fluorescent chemical – which emits light under certain conditions
how do dna probes work
How do DNA probes work…?
  • The probe contains bases which are complimentary to the DNA sequence you want to find…
  • The DNA being tested is heated to separate the strands…
  • The single-stranded probe is mixed with the denatured DNA…
  • What then happens?
  • The probe hybridizes to the gene you want to find
  • The site where the probe binds can then be identified using…?
  • …the radioactivity or fluorescence that the probe emits
dna sequencing
DNA sequencing
  • But, how do we know what nucleotides / bases to make the probe out of?
  • We need find out the sequence of nucleotides / bases in the gene you are trying to locate…
  • Various methods…
  • E.g. Sanger method
  • Remember: Sanger used for sequencing
sanger method of dna sequencing
Sanger method of DNA sequencing
  • Uses modified nucleotides that cannot attach to the next base in the sequence…
  • These are know as Terminators…
  • They end the synthesis of a DNA strand
  • Four different terminator nucleotides are used…
  • Why four?
  • One containing Adenine, one with Thymine, one with Guanine, one with Cytosine…
sanger method continued
Sanger method continued…
  • Set up four tubes, each containing…
  • Many single-stranded fragments of the DNA to be sequenced
  • A mixture of nucleotides, representing each of the 4 bases
  • A small amount of ONE of the four terminator nucleotides
    • Test tube 1 = adenine terminator nucleotide
    • Test tube 2 = thymine terminator nucleotide etc, etc
  • A primer, labeled with a radio-isotope or a fluorescent dye
  • DNA polymerase
  • Why is a primer needed?
  • To start DNA synthesis
  • Why is DNA polymerase needed?
  • To catalyse DNA synthesis once it has begun
sanger method continued1
Sanger method continued…
  • The binding of nucleotides to the DNA fragments is random, so…
  • …the binding of a normal nucleotide is as likely as the binding of a terminator nucleotide.
  • So how long will the newly synthesized DNA be?
  • Depending where the terminator nucleotide binds on the DNA fragment…
  • …DNA synthesis may be terminated after a few nucleotides (so, short) , or…
  • …after a long fragment has been synthesized.
sanger method continued2
Sanger method continued…
  • Therefore the DNA fragments in each test tube will be of varying lengths.
  • But they all have one thing in common…
  • …all the fragments of new double-stranded DNA in a particular test tube will each end with a nucleotide that has the same base e.g A in tube 1, T in tube 2 etc.
  • How can these fragments be identified?
  • Because of the labeled primer
  • The fragments now need to be separated…
sanger method continued3
Sanger method continued…
  • DNA has a negative charge. Why?
  • Because of all the phosphate groups
  • Therefore DNA can be separated using…
  • …Gel Electrophoresis
  • The DNA fragments are applied to agar gel
  • A voltage is applied across the gel
  • Which fragments move the furthest / least far?
  • The smallest / the biggest
  • A photographic plate is laid on the gel for several hours. Why?
  • The Radiolabeled fragments expose the film and so reveal their location
interpreting the results

These are the terminator nucleotides

Interpreting the results…
  • From where should you read the results?
  • From the top down
  • Why?
  • Because the shortest fragments move the longest distance.
  • So, what is the first base in the fragment?
  • C
  • Then what ?
  • T
  • What’s the full sequence?
  • What’s the sequence of the gene (not the DNA you’ve just made)?
  • ie. The complimentary base sequence!

Short fragments of DNA

Increasing distance from the origin

Long fragments of DNA

video clips
Video clips
  • http://www.youtube.com/watch?v=aPN8LP4YxPo

Explains the process nicely.

  • http://www.youtube.com/watch?v=6ldtdWjDwes

Does not explain the process very well but shows how you arrive at the final sequence.

restriction mapping
Restriction mapping
  • Sanger sequencing can only be used for fragments of DNA up to 500 bases long.
  • Before sequencing, larger genes and entire genomes must first be cut into smaller fragments. How?
  • Using restriction endonucleases
  • The fragments must then be separated. How?
  • Using gel electrophoresis
  • And then each fragment is sequenced separately
  • Then what?
  • The sequenced fragments have to be pieced back together. This is called restriction mapping
restriction mapping uses
Restriction mapping uses…
  • Restriction endonucleases, which cut DNA at recognition sites
  • The DNA to be sequenced is cut using a series of different endonucleases.
  • How can the distance between the recognition sites can be determined?
  • By the pattern the fragments make on the gel
  • An example is explained on page 269 of Nelson Thornes
restriction mapping1
Restriction mapping
  • E.g… of a plasmid with 100,000 bases (100kb)…
  • Plasmids are circular, so if only one restriction endonuclease were used what would be the product?
  • A single piece of DNA which is 100kb long, regardless of which enzyme were used.
  • But what if more than one restriction enzyme were used?
  • There will be two cuts to the plasmid and so two different length fragments will be produced
  • Always?
  • It is possible that the two enzymes make cuts 180degrees around the plasmid from each other, so that each fragment is exactly half of 100kb i.e. 50kb

Possible gel electrophoresis results of 3 different restriction mapping “double digests”

The numbers are the lengths of the fragments produced by the enzymes in kilobases

  • Why do the numbers in each column add up to 100?
  • Because the original plasmid was 100kilobases big
  • If the table was a gel, in what direction did the fragments move?
  • From top to bottom (10kb migrates further than 90kb)
restriction map for an e coli cloning vector plasmid
Restriction map for an E. coli Cloning Vector Plasmid
  • This restriction map shows the recognition sites of various endonucleases.
  • The map also shows the antibiotic resistance marker genes.
  • Genes to be cloned can be inserted into numerous places depending on the restriction endonuclease used
  • Look at the positions of BamHI and HindIII…
  • Assuming the plasmid was 100kb, what size fragments would a double digest with these two enzymes produce?
  • 10kb & 90kb…
  • Admittedly the delivery is fairly boring…
  • But it is spot on in terms of content!
  • http://www.youtube.com/watch?v=c97VqOJkQ88

Video shows how to use the results of restriction mapping.

  • Both DNA sequencing an restriction mapping are now routinely automated and…
  • computers are used to analyse the results.
  • The machines use…
  • 4 different fluorescent dyes to label the terminators, one for each kind (radiolabeled primers not used)
  • The DNA synthesis occurs in a single test tube (not 4)…
  • And PCR is used to speed it up.
  • The electrophoresis occurs in a narrow capillary gel
  • The results are red by lasers
  • Also, prior to digestion with endonucleases, PCR is used to provide the fragments needed at the start
  • Think: Cost Benefit Analysis? Expensive but quick, accurate
  • What is a DNA probe?
  • State TWO roles of a primer used in the Sanger method of DNA sequencing
  • Describe gel electrophoresis?
  • What is meant by the term “restriction map”
  • List the differences between the original manual method of restriction mapping, and the current automated version
implications of genetic screening
Implications of Genetic Screening
  • With information comes power, opportunity and responsibility…
  • Who decides who should be screened? It’s expensive and budgets are limited
  • Who has access to the test results? Employers? Insurers? Lenders?
  • Does a carrier have a responsibility for the alleles they pass on? Note – the genetic disease Tay-Sachs involves constant pain and death at the age of four years
  • Does mankind have a responsibility to maintain genetic diversity? Should we preserve mutant genes for the sake of human evolution?
  • Who decides what is a defect? Tay-sachs, yes, but ginger hair…?