biotechnology: DNA and RNA anaylis

Biotechnology

use of basic science from the feild of biology to solve human problems

DNA/RNA analysis

takes a sample of DNA and learn about it

PCR

Gel Electrophoresis

Sequencing

DNA manipulation

Gene cloning

CRISPR

Why is DNA/RNA anyslis risky and how is it overcome

Forensic sceintits have little DNA avaible and if Analysis fails then there is no DNA left

To combat this they make copies of the DNA

Polymerase Chain Reaction (PCR)

Makes more copies of the DNA

DNA replciation in a test tub

Ingredients in PCR

Crim scene DNA

Primers

DNA polymerase

Nucleotides

Buffer/cofactors

3 steps of PCR

1. Denaturation

2. Annealing

3. Elongation

Denaturation

Mimics helicase in DNA replication

Heated to 95 degrees

Hydrogen bonds and broken and DNA is speerated into 2 strands

Annealing

Sample is cooled (55 C) and primers bind

Primers are DNA based

Elongation

72 C

DNA polymerase uses the free -OH group at the end of the primers to crete two new DNA strnads

Is PCR reusable

Yes! PCR can be cycled again and again this time replicating the newly made DNA strands

30 cycles of PCR significance

Single strand can be replicated into more than a billion copies

Thermocycler

Machine for PCR

Will cycle through the temperatures needed for PCR to repeat the process over and over again

DNA sequencing

Allows scientists to determine the exact order of nucleotides in a given DNA sample

• Can be preformed to short DNA molecules as well as an organisms entire geonone

DNA sequencing purpose

Can detect mutations

Useful for detecting small changes in DNA that have big consequences

• Can detect 3 nucleotides in a gene that codes for a chloride transport protein that results in Cystic Fibrosis

RNA sequencing

What protiens are being made (cancer detection)

Preformed in a similar manner to DNA sequencing but reveals changes in gene expression

mRNA from cells can be collected and sequenced to determine if gene expression has changed

RNA sequencing steps

1. mRNA are isolated from tissue being studid

2. mRNA are cut into simular sized smaller fragments

3. mRNAs are reverse transcribed into cDNAs of the same size

4. cDNAs are sequenced

5. Short sequences are mapped by computer onto the genome sequence

cDNA and stability

cDNA is more stable than RNA

restriction enzymes

special class of enzymes that cut DNA at specific sequences

significane of restriction enzymes

Cut the DNA into diffrent sized peices or fragments

Different combination of fragment sizes is unique to each person

Gel Electrophoresis

A type of sceincy machine that can comapre the DNA fragments of diffrent sizes and line them up

Gel electrophoresis Steps

1. Crime scene DNA is copied and then cut with restriction enzymes

2. DNA from potential suspects is collected and cut with the same restriction enzyme

3. RNA samples are loaded into a gel block

4. Run through gel electrophoresis machine

5. DNA spreads out due to the size of bands

   1. Large fragments travel slower through the gel, while smaller fragments travel faster.

6. Each person produces a unique banding pattern in gel electrophoresis

Gel electrophoresis image