human bio evidence for evolution - LESSON 1 DNA SEQUENCING AND PROFILING TECHNIQUES

how do ddNTP and dNTP differ?

dideoxynucleotide triphosphate lacks a hydroxl group which is needed for further attachments.

dna sequencing

determines the sequences of nucleotide bases in a DNA sample, using special nucleotides calls ddNTP

what happens when a dideoxynucleotide is added to a forming strand of DNA

elongation of that strand is ended. the next deoxynucleotide is not able to bond onto the strand

6 requirements for DNA sequencing

• single stranded dna

• free nucleotides (ATGC)

• dideoxynucleotides labelled with different coloured fluorescent dye for easy detection

• primers

• taq dna polymerase

• dna sequencing machine

7 steps of dna sequencing

1. DNA is extracted from cell nucleus and broken up into multiple pieces using enzymes

2. multiple copies of this DNA are produced

3. double stranded DNA separated into single strands

4. primers added which bind to one strand and initiate the replication process

5. the second strand of DNA is recreated by adding complimentary nucleotides (dNTP) in correct sequence using taq DNA polymerase

6. a terminator nucleotide (ddNTP) stops the nucleotide sequence

7. the strands are compared to determine the nucleotide sequence

what are primers in dna sequencing

segments of dna complimentary to the target sequence of dna and initiates replication by dna polymerase

uses of dna sequencing

• it can detect change alleles (mutations) and will show whether an individual has a particular disease, eg cystic fibrosis or sickle cell anaemia. this enables people to seek effective treatment, and possibly even prevent the disease or lessen the effects.

• used for maternity and paternity tests

gel electrophoresis

• dna samples are loaded into wells at one end of an agarose gel.

• when current is run through the cell and the buffer it sits in, the DNA fragments move through the gel from the negative terminal to the positive terminal (sugar phosphate backbone is negatively charged and opposites attract)

where do larger dna fragments go in gel electropheresis

they move slower and are found near the top (neg terminal)

where do smaller dna fragments go in gel electropheresis

they move faster and are found near the bottom (pos terminal)

why are markers run in gel electrophoresis

the markers have set fragment sizes and are run to compare the unknown fragments to determine their sizes

3 advantages in DNA profiling

• DNA lasts a long time in the environment (eg dried blood lasts 3-4 years)

• only requires a very small amount of tissue

• is individual to each person and can be used to identify relationships

why is non coding/ junk dna used in dna profiling

as the dna usually consists of many highly repetitive sequences of bases. the number of times the sequences are repeated is subject to mutation and therefore unique to an individual organism

3 uses of dna profiling

• crime/forensics

• tracing ancestry

• indentifying hereditary diseases (probability of child having disease, early detection of diseases)