Investigatory Project Biology

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INVESTIGATORY PROJECT

BIOLOGY

NAME: TUSHAR GAUTAM

CLASS: 12THA

ROLL NO.: 13

SCHOOL: R.P.V.V. SEC-10 DWARKA NEW DELHI-11007

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DNA Fingerprinting

Unless they are identical twins, individuals haveunique DNA

DNA !"#$%&'&"#("#$

The name used for the unamiguous identifying techniquethat ta!es advantage of differences in DNA sequence

The process of DNA fingerprinting egins y isolatingDNA from

lood, semen, vaginal fluids, hair roots, s!in, s!eletalremains, or elsewhere

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"olymerase #hain $eaction %"#$&

'f there is only a small amount of DNA availale for DNA

Fingerprinting

augment the amount of DNA using a

technique called PCR

PCR is doing DNA replication in a test

tube

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Template

and coolto anneal

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Primer

PrimerTemplate

Like ALL DNApolymerases 53

Taq polymerase canonly add to the 3 end

of an existing

nucleotide

A DNA primer that is

complementary to the

template is used to

supply that 3 end

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DNA Fingerprinting

After we isolate the DNA and amplify it with "#$ Treat the DNA with &%)(&"*("+# %#,%)

cut DNA at specific sequences

(veryone)s DNA is different, so everyone)s DNA will cut at different sites

This results in different si*ed fragments

The different si*ed fragments are called &%)(&"*("+# !&/$%#(%#$( '++&'")), or RLP)

+e can oserve the different si*ed fragments in an eperiment

that separates DNA ased on fragment si*e called -el(lectrophoresis

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$F." Analysis

(veryone has geneticsequences called/&"/4% #54%&

(/#6% &%'%/(), orVNTR)

(veryone has differentamounts of /NT$s

The /NT$s ma!e the

different si*ed $F."s

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-el (lectrophoresis

Fragments of DNA from restriction en*yme cleavageare separated from each other when they migratethrough a support called an /$/&+)% $%

't is similar to the yummy food 0ell12 gelatin

't is actually made out of some of the same ingredients

The si*e1ased separation of 3olecules of DNAseparate ased on si*e when an electric current isapplied to an agarose gel

This is $% %%*(&+'+&%)")

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-el (lectrophoresis

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-el (lectrophoresis

The separated DNA fragments are then drawn out of

the gel using a nylon memrane

The nylon memrane is treated with chemicals that

rea! the hydrogen onds in DNA and separate thestrands

The single stranded DNA is cross lin!ed to the nylonmemrane

4y heat or U/ light

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-el (lectrophoresis

'ncuate the nylon memrane with a radioactive '&+4%of single stranded DNA complementary to the

/NT$s

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-el (lectrophoresis

-el (lectrophoresis

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The radioactive proe shows up on photographicfilm

4ecause as it decays it gives off light

The light leaves a dar! spot on the film

Different individuals have different patterns ofands these ma!e up the fingerprint

-el (lectrophoresis

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This rotocol is kno!n as "outhern #lotting5outhern 4lotting

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DNA Fingerprinting

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DNA fingerprints can e used to determine whichone fragments elong to which individual

DNA Fingerprinting

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DNA fingerprints of children should e similar to the thoseof parents

DNA fingerprinting can show which individuals are theparents of specific children

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Northern 4lot Analysis

Northern lotting analy*es $NA much the sameway that 5outhern lotting does DNA6

$NA is etracted from the cell, undergoes gelelectrophoresis, and is ound to a filter7

8yridi*ation etween ound cellular $NA and a laeledproe occurs7 The si*es of the $NA fragments detectedy the proe can e determined

Northern 4lot Analysis

Northern lot analysis is used for determining6

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The si*e%s& of m$NA encoded y a gene7 Northern lotshave shown that different m$NA species arise from the

same region of DNA, suggesting differential use ofpromoters and terminators, and9or alternative m$NAprocessing7

+hether a specific m$NA is present in a cell type, and ifso, at what levels7 -ene activity is measured in this way,

and $NA sampling is widely used to study development,tissue speciali*ation, or the response of cells to variousphysiological stimuli7

"roducing $ecominant "roteins

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The first step in the production of rBGHprotein %or insulin&is

to transfer the BGHgene %or human insulin gene& from the nucleus ofa cow cell %or human cell& into a acterial cell

8ow do we do that::::

; steps are involved in turning a cow BGHgene into arecominant 4-8 %r4-8& gene in a acterial cell

rBGHgene means that this product is genetically engineered

with the rindicating recominant

"roducing $ecominant "roteins

< steps are involved in turning a cow BGHgene into arecominant 4-8 %r4-8& gene in a acterial cell

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1. 3a!e lots of copies of the cow 4-8 gene in the la in atest tue

2. #ut cow 4-8 gene with restriction en*ymes

3. 'nsert this cow 4-8 gene into acterial DNA = r4-8

4. 'n>ect the acterial DNA containing the r4-8 into acteria

5. -row up lots of these genetically engineered acteria andpurify the r4-8 cow protein they are ma!ing

C+#"#$ / G%#% U)"#$ B/*(%&"/

5tep ?7 3a!e lots of copies of the 4-8 -ene

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Use "#$ to amplify only the cow 4-8 gene fromthe cow chromosomes

$ememer, "#$ is >ust replicating DNA in thelaoratory in a test tue

(nd up with lots and lots of copies of the cow 4-8gene DNA in a test tue

#loning a -ene Using 4acteria

5tep @7 "repare the cow 4-8 -ene for inserting intoacterial DNA

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The cow 4-8 gene ends are sliced using&%)(&"*("+# %#,%)

$estriction en*ymes cut DNA only at specificsequences that leave the doule1stranded DNA>agged or stic!yB on the ends

$estriction en*ymes cut the DNA in a staggered

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pattern, leaving sticky ends

Restrictin en!"#e c$t

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Fig. 16.2Examples of how

differentrestriction

enzymes cleaveDNA

eter !. "#ssell$ iGenetics% &opyright ' earson Ed#cation$ (nc.$p#)lishing as *en+amin mmings.

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#loning a -ene Using 4acteria

5tep ;7 'nsert the BGH-ene into the 4acterial"lasmid

The acterial plasmid is also cut with the restrictionen*yme, leaving stic!y ends

A plasmid is a small circular DNA that is separate fromthe acterial genome

5tic!y ends of the cut 4-8 DNA attach ycomplementary ase pairing to the stic!y ends of

the cut plasmid DNA

This is now recominant DNA

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Fig. 16.,&leavage of DNA )y the restriction enzyme Eco"(

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eter !. "#ssell$ iGenetics% &opyright ' earson Ed#cation$ (nc.$ p#)lishing as *en+amin mmings.

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#loning a -ene Using 4acteria

5tep C7 'nsert the $ecominant "lasmid into a 4acterial #ell The recominant plasmid containing the r4-8 is then placed into

acterial cells

5tep

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TaDa $ecominant proteins

C+#"#$ / G%#% U)"#$ B/*(%&"/

8ow can acteria produce the cow 4-8 protein:

This wor!s ecause acteria use the same geneticcode as cows %and all living things&

2ther proteins are made in this way6

'nsulin for diaetics

#lotting factors for hemophiliacs

#ancer treatment drugs

#loning a -ene Using 4acteria

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Using the lacEgene as areporter of

geneepression

$eporter gene proteinencoding genewhose

epression in

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the cel